This study was supported by the U.S. National Institutes of Health (NIH) grants R01HL090767, R01HL090767-02S1, P30 DK06010, and The NepCure Foundation Established Investigator New Direction Grant, (to A.S.-U.).

1. Seeding Cells
<ol>
	<li>Pretreat 24 mm filters with 10% collagen I (prepare 10% collagen I in Minimal Essential Medium (MEM), cover the entire surface of the filter with the collagen solution, incubate under the UV light at room temperature for 30 min, and in a cell culture incubator at 37 &#186;C for 1 hr, suction off the excess collagen after incubation).</li>
	<li>Prepare cell culture medium (MEM gassed with CO2 for 20 min, 10% Fetal Bovine Serum (FBS), 50 U/ml penicillin, 50 U/ml streptomycin, 2 mM L-glutamine, 0.5 &#956;g/ml puromycin)</li>
	<li>Seed CFBE41o- cells on six 24 mm filters for endocytosis and recycling, respectively, at 1 x 106/filter.</li>
	<li>Remove the apical medium the day after seeding and feed daily from the basolateral side only.</li>
	<li>Feed with selection antibiotic negative medium 24 hr before the experiment. Perform experiment in CFBE41o- cells 6-10 days after seeding.</li>
</ol>
2. Preparations Before the Experiment (Similar for the Endocytosis and Recycling Assay)
<ol>
	<li>Set up a bench space in the cold room. Endocytic and recycling assays should be performed in the cold room except for the incubation at 37 &#176;C. Plates containing the 24 mm filters should be placed directly on the bench top in the cold room.</li>
	<li>Turn on the 37 &#186;C incubator.</li>
	<li>Prepare 500 ml of PBS++, pH 8.2 (Dulbecco&#8217;s Phosphate Buffered saline (PBS), 1 mM magnesium chloride, and 0.1 mM calcium chloride, pH 8.2) and keep 250 ml at 37 &#176;C in an incubator and 250 ml at 4 &#176;C in the cold room.</li>
	<li>Fill wells in a 6&#160;well plate&#160;with PBS++, pH 8.2, 37 &#176;C and keep in the incubator at 37 &#176;C.</li>
	<li>Fill wells in another 6&#160;well plate with PBS++, pH 8.2, 4 &#176;C and keep in the cold room at 4 &#176;C.</li>
	<li>Prepare 100 ml of PBS++, pH 8.6 at 4 &#176;C and keep in the cold room.</li>
	<li>Prepare biotin containing the disulfide bond and NHS ester at a concentration 0.8 mg/ml in PBS++, pH 8.2, 4 &#176;C within 30 min of the biotinylation step (the volume of biotin buffer should cover completely the entire surface of the filter); we recommend 1.5 ml/24 mm filter.</li>
	<li>Prepare 100 ml&#160;of GSH buffer in water, pH 8.6 and cool to 4 &#186;C (75 mM sodium chloride, 1 mM magnesium chloride, and 0.1 mM calcium chloride, 50 mM GSH, 80 mM sodium hydroxide, and 10% FBS). GSH and sodium hydroxide should be added just before the experiment. Check the pH and adjust to 8.6; sodium hydroxide neutralizes the carboxyl groups and deprotonates half the cysteine residues in glutathione. It is strongly buffered at the pKa of this cysteine, which is 8.64. (Prepare the same volume of GSH buffer for the recycling assay).</li>
	<li>Prepare 50 ml of Lysis buffer, pH 8.2 and keep at 4 &#176;C (25 mM HEPES, pH 8.2, 1% (v/v) Triton X-100, 10% (v/v) glycerol); add Complete protease inhibitor cocktail per 50 ml of lysis buffer and cool to 4 &#186;C; check the pH after adding Complete as a drop in pH may occur.</li>
	<li>Prepare Laemli sample buffer with 100 mM DTT.</li>
	<li>Prepare 1x Running buffer (100 ml of 10x Running buffer, 900 ml of water).</li>
	<li>Prepare 1x Transfer buffer and cool to 4 &#186;C (100 ml of 10x Transfer buffer without sodium dodecyl sulfate (SDS), 200 ml of methanol, 700 ml of water).</li>
</ol>
3. Endocytic Assay
CFTR polarizes to the apical membrane domain; thus, the protocol describes biotinylation of the apical membrane domain. Biotinylation of the basolateral membrane domain will be required to study endocytosis of proteins polarizing to the basolateral membrane. 
Workflow: Biotinylation of cell surface proteins at 4 &#186;C &#8594; Warming to 37 &#186;C to load endocytic vesicles with biotinylated proteins &#8594; Cooling to 4 &#186;C to stop endocytic trafficking &#8594; Reduction of the disulfide bond in biotin attached to proteins that have remained at the cell surface &#8594; Cell lysis &#8594; Isolation of biotinylated (i.e. endocytosed) proteins with streptavidin agarose &#8594; Elution of biotinylated proteins from streptavidin agarose &#8594; Protein electrophoresis and western blotting.
<ol>
	<li>Bring the plate containing six 24 mm filters (Table 1: samples a-c) from the cell culture incubator and transfer quickly to the plate filled with cold (4 &#186;C) PBS++, pH 8.2 on ice.</li>
	<li>Let PBS++ overflow the apical surface to cover quickly the entire surface. Bring the plate on ice to the cold room and set on the bench top.</li>
	<li>Remove PBS++ quickly by turning the plate upside down holding filters in place to prevent falling out. Add ~2 ml of PBS++ to the apical and basolateral side.</li>
	<li>Wash filters with ~2 ml of cold PBS++, pH 8.2&#160;3x for 2 min. Suction off the wash from one side at a time, apical or basolateral and add fresh PBS++, pH 8.2 to one side at a time.</li>
	<li>Suction off the last wash and add 1 ml of PBS++, pH 8.2 to the basolateral side. Add 1.5 ml of the biotin buffer to the apical side and incubate in the dark for 30 min. Avoid spilling buffers to the contralateral side.</li>
	<li>Suction off the biotin buffer from the apical side and wash with PBS++, pH 8.2 3x for 2 min.</li>
	<li>Set aside in a different plate two filters (Table 1: samples a and b), add ~2 ml of PBS++, pH 8.2 to the apical and basolateral side and keep in the cold room. Keep the remaining 4 filters in one plate (Table 1: samples c) and add ~2 ml of PBS++ pH 8.2 on the apical and basolateral side of each filter. Place the plate on ice and bring to the bench top next to the 37 &#186;C incubator.</li>
	<li>Transfer quickly filters from ice to the plate in the 37 &#186;C incubator filled with prewarmed PBS++ pH 8.2 and incubate one filter each precisely for 2.5, 5.0, 7.5, or 10 min covering the entire surface of each filter with warm PBS++ pH 8.2.</li>
	<li>During the incubation fill completely four wells in the plate on ice with cold (4 &#186;C) PBS++ pH 8.2.</li>
	<li>After the indicated time of incubation, turn each filter upside down to drain the warm (37 &#186;C) PBS++, pH 8.2 and transfer to the plate on ice. Add cold PBS++, pH 8.2 to overflow the apical side and cover the entire surface of each filter quickly.</li>
	<li>After transferring filters to ice bring the plate to the cold room and set on the bench top.</li>
	<li>Suction off PBS++ from both sides in all filters (samples a-c) and fill the basolateral side with 1 ml of PBS++ pH 8.6. Add 1.5 ml of PBS++, pH 8.6 to the apical side of the filter a (Table 1: sample a).</li>
	<li>Reduce the disulfide bond in biotin attached to the apical membrane proteins with the GSH buffer (pH 8.6) in filters b and c (Table 1: sample b and c).</li>
	<li>Add 1.5 ml of the GSH buffer to the apical side, incubate for 15 min, and repeat six times.</li>
	<li>Keep 1 ml of PBS++ pH 8.6 on the basolateral side. Avoid spilling the buffer to the basolateral side.</li>
	<li>After completing the procedure 3.15, wash all filters (Table 1: samples a, b, and c) briefly with PBS++ pH 8.2 2x, suction off the wash from the apical and basolateral side and set the plate with filters at a 45&#176; angle to drain all wash and suction again.</li>
	<li>Place the plate flat on the bench top and add 500 &#956;l of lysis buffer to the apical side of each filter and incubate in the cold room shaking for 15 min.</li>
	<li>Scrape cells with a rubber policeman and collect in a 1.5 ml Eppendorf tube.</li>
	<li>Spin at 14,000 x g for 10 min at 4&#160;&#176;C.</li>
	<li>Transfer the supernatants to fresh 1.5 ml tubes and discard the pellets. The supernatants are now called whole cell lysates (WCL).</li>
	<li>Add 100&#160;&#956;l of Laemli sample buffer with DTT to 50 &#956;l of WCL (10% of the entire WCL) and incubate at 37 &#176;C for 30 min&#160;(WCL samples).</li>
	<li>Prepare 200 &#956;l aliquots of 50% streptavidin agarose and remove all wash buffer with a 27 G needle attached to a suction: wash 2x with 1 ml of PBS, pellet beads with pulse spin between the washes; wash once with 1 ml of&#160;lysis buffer to equilibrate before adding WCL; suction dry the agarose prior to adding WCL.</li>
	<li>Incubate the remaining WCL with washed streptavidin agarose (bring the volume up to 1.2 ml with lysis buffer and rotate in the dark for at least 2 hr or O/N in the cold room).</li>
	<li>Wash the streptavidin agarose&#8211;biotinylated protein complexes three times with 1 ml of lysis buffer (the lysis buffer does not need to contain Complete during washing), pulse spin the agarose between washes,&#160;and suction dry after the last wash.</li>
	<li>Incubate the streptavidin agarose&#8211;biotinylated protein complexes with 65 &#956;l Laemli sample buffer with DTT at 85 &#176;C for 5 min.</li>
	<li>Pulse spin and collect the eluted biotinylated protein complexes.</li>
	<li>Load 7.5% gels with 40 &#956;l of the WCL samples and the entire volume of the biotinylated (BT) samples per well.</li>
	<li>Run gels at 120 V in SDS containing running buffer.</li>
	<li>Transfer proteins to a PVDF membrane at 90-95 V for 1.5 hours (as long as &#60;400 A) in a transfer buffer without SDS.</li>
	<li>Block in 5% nonfat milk in TBS-T 0.1% O/N. Blot both membranes with anti-CFTR mouse monoclonal antibody CFF596 and a goat anti-mouse HRP-conjugated secondary antibody.</li>
	<li>Perform chemiluminescence. Reblot both membranes with either anti-ezrin or anti-actin antibody.</li>
</ol>
4. Recycling Assay
Workflow: Biotinylation of cell surface proteins at 4 &#186;C &#8594; Warming to 37 &#186;C to load endocytic vesicles with biotinylated proteins &#8594; Cooling to 4 &#186;C to stop endocytic trafficking &#8594; Reduction of the disulfide bond in biotin attached to proteins that have remained at the cell surface &#8594; Warming to 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the cell surface &#8594; Cooling to 4 &#186;C to stop endocytic trafficking &#8594; Reduction of the disulfide bond in biotin attached to proteins that have recycled to the cell surface &#8594; Cell lysis &#8594; Isolation of biotinylated proteins (i.e. those that have not recycled) with streptavidin agarose &#8594; Elution of biotinylated proteins from streptavidin agarose &#8594; Protein electrophoresis and western blotting.
<ol>
	<li>Biotinylate the apical surface proteins in five 24 mm filters (Table 2: samples a-d) following the procedure described in the Endocytic assay (section 3.1-3.6).</li>
	<li>In the cold room, set in a new plate two filters (Table 2: samples a and b), add ~2 ml of PBS++, pH 8.2 to the apical and basolateral side and keep in the cold room.</li>
	<li>Keep the remaining 3 filters in one plate (Table 2: samples c and d) with ~2 ml of PBS++ pH 8.2 on the apical and basolateral side of each filter. Place the plate with filters c and d on ice and bring to the bench outside of the 37 &#186;C incubator.</li>
	<li>Transfer filters quickly from the plate on ice to the plate filled with prewarmed PBS++ pH 8.2 in the incubator and keep for 5.0 min.</li>
	<li>During the incubation fill 3 wells of the plate on ice with cold (4 &#186;C) PBS++ pH 8.2.</li>
	<li>Transfer filters after the 5.0&#160;min&#160;incubation at 37 &#186;C to the plate on ice. Let the cold PBS++ pH 8.2 to overflow the apical side and cover the entire surface of each filter quickly.</li>
	<li>Bring the plate on ice to the cold room and set on the bench top.</li>
	<li>Suction off PBS++ pH 8.2 from both sides of filters a, b, c, and d and add 1 ml of PBS++ pH 8.6 to the basolateral side.</li>
	<li>Keep filters a and b separately from filters c and d. Add 1 ml of PBS++ pH 8.6 to the apical side of filters a and b.</li>
	<li>Reduce the disulfide bond in biotin remaining at the cell surface in filters b, c and d following the procedure described in the endocytic assay (steps 3.13-3.15).</li>
	<li>Wash filters b, c, and d briefly with PBS++ pH 8.2 2x and replace with fresh PBS++, pH 8.2. Place filters d in a new plate and bring on ice to the bench top outside the 37 &#186;C incubator.</li>
	<li>Transfer quickly filters from the plate on ice to the plate in the incubator filled with prewarmed PBS++ pH 8.2 and incubate one filter each precisely for 2.5 or 5.0 min as described above in steps 4.4-4.9.</li>
	<li>Reduce the disulfide bond in biotin attached to the apical membrane proteins with the GSH buffer after the second incubation at 37 &#186;C in filters d as described in 4.4 with the exception that only three 15&#160;min&#160;incubations with the GSH buffer will be done during this step. Keep filters a, b, and c in PBS++ pH 8.6 on the apical and basolateral side during this step.</li>
	<li>For the cell lysis, and Western blotting follow procedures described in the endocytic assay (steps 3.16-3.31).</li>
</ol>

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Cell Biol. 10, 597 (2009).</li><li>Bretscher, M. S., Lutter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+detecting+endocytosed+proteins.">A new method for detecting endocytosed proteins.</a> EMBO J. 7, 4087 (1988).</li><li>Watts, C., Davidson, H. 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Cell Physiol. 283, C489 (2002).</li><li>Swiatecka-Urban, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDZ-domain+interaction+controls+the+endocytic+recycling+of+the+cystic+fibrosis+transmembrane+conductance+regulator.">PDZ-domain interaction controls the endocytic recycling of the cystic fibrosis transmembrane conductance regulator.</a> J. Biol. Chem. 277, 40099 (2002).</li><li>Swiatecka-Urban, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+VI+Regulates+Endocytosis+of+the+Cystic+Fibrosis+Transmembrane+Conductance+Regulator.">Myosin VI Regulates Endocytosis of the Cystic Fibrosis Transmembrane Conductance Regulator.</a> J. Biol. 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Physiol. 290, C862 (2006).</li><li>Swiatecka-Urban, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+Vb+is+required+for+trafficking+of+the+cystic+fibrosis+transmembrane+conductance+regulator+in+Rab11a-specific+apical+recycling+endosomes+in+polarized+human+airway+epithelial+cells.">Myosin Vb is required for trafficking of the cystic fibrosis transmembrane conductance regulator in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells.</a> J. Biol. Chem. 282, 23725 (2007).</li><li>Ye, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-Cbl+facilitates+endocytosis+and+lysosomal+degradation+of+cystic+fibrosis+transmembrane+conductance+regulator+in+human+airway+epithelial+cells.">c-Cbl facilitates endocytosis and lysosomal degradation of cystic fibrosis transmembrane conductance regulator in human airway epithelial cells.</a> J. Biol. Chem. 285, 27008 (2010).</li><li>Cihil, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disabled-2+protein+facilitates+assembly+polypeptide-2-independent+recruitment+of+cystic+fibrosis+transmembrane+conductance+regulator+to+endocytic+vesicles+in+polarized+human+airway+epithelial+cells.">Disabled-2 protein facilitates assembly polypeptide-2-independent recruitment of cystic fibrosis transmembrane conductance regulator to endocytic vesicles in polarized human airway epithelial cells.</a> J. Biol. Chem. 287, 15087 (2012).</li><li>MacEachran, D. 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The authors declare that they have no competing financial interests.

The success of endocytic and recycling assays depends on efficient biotinylation of CFTR at the plasma membrane. Biotin has to be prepared immediately before use because the NHS-ester moiety readily hydrolyzes and becomes nonreactive. Moreover, the biotinylation step requires strict temperature control at 4 &#186;C to stop protein trafficking. If the temperature is raised during the biotinylation step above 4 &#186;C protein trafficking may occur resulting in the biotinylation of variable amounts of CFTR.
The success of endocytic and recycling assays also depends on efficient reduction of the disulfide bond in the biotinylated CFTR remaining at the plasma membrane.&#160;&#160; The amount of CFTR remaining biotinylated after the GSH treatment should be &#60;10% of the total biotinylated CFTR at time zero. Experiments where the amount of biotinylated CFTR is &#62;10% in the GSH control sample should be excluded. Figure 1D&#160;demonstrates &#8220;failed&#8221; experiment where the amount of biotinylated CFTR in the GSH control was 14.5%. Insufficient reduction of the disulfide bond in the biotinylated CFTR may be secondary to poor control of temperature during the experiment. If the temperature is raised during the GSH treatment above 4 &#186;C protein trafficking may occur and the biotinylated CFTR endocytosed as a result of the unintentional increase in temperature will increased the background CFTR. Alternatively, insufficient reduction of the disulfide bond in the biotinylated CFTR may result from decreased half-life of GSH in aqueous solution caused by oxidation in air or from inappropriate pH. As previously determined GSH remains strongly buffered at pH 8.64. In our experience six 15&#160;min treatments with the GSH buffer reduced the disulfide bond by more than 90%, more efficiently than one 90 min treatment.
The cell culture requires special attention. Biotin has to reach the plasma membrane domain in intact cells where the protein of interest is expressed. We have used the biotinylation based endocytic and recycling assays to study endocytosis and recycling of CFTR expressed in the apical membrane domain. These assays may be also used to study endocytic trafficking of proteins located in the basolateral membrane domain in cells cultured on semipermeable growth supports. Epithelial cells that form multilayers are not suitable for the these assays as biotin and GSH buffers will only reach the top layer of cells to detect the apical membrane proteins or the bottom layer to detect the basal membrane proteins. Epithelial cells cultured on plastic may be more prone to washing off during the assays. Random loss of cells during the experiment will compromise results. We recommend periodic examination of the monolayer integrity under a microscope in experiments where cells are cultured in plastic tissue culture dishes. Coating of tissue culture dishes with collagen may increased cell adherence.
The biotinylated protein samples should be routinely tested for contamination with intracellular proteins. Detection of intracellular proteins in the biotinylated protein samples may indicate insufficient washing of the streptavidin agarose&#8211;biotinylated protein complexes after incubation with WCL. Thus, the first step is to increase the washing efficiency. Moreover, presence of cells with compromised plasma membrane integrity will allow access of biotin to intracellular proteins. Western blotting for a protein expressed exclusively in an intracellular compartment such as the endoplasmic reticulum could be used to detect biotinylation of intracellular proteins. Alternatively, cytoskeletal proteins such as actin or ezrin may be used to test for contamination of the biotinylated protein samples with intracellular proteins. Cytoskeletal proteins may form complexes with transmembrane proteins and small amounts of the cytoskeletal proteins may be detected in the biotinylated protein samples. However, in our experience, the ratio of biotinylated/WCL ezrin or actin is &#60;1:1,000 and thus, these proteins are suitable for determination of cell membrane integrity in the biotinylation-based assays.
The biotinylation reaction can be quenched with glycine or Tris to remove nonreacted biotinylation reagent as previously reported17. Moreover, GSH can be quenched with iodoacetamide6. In our experience the nonreacted biotinylation reagent and GSH can be efficiently removed by gentle washing with PBS++ without the use of quenching reagents.
The choice of streptavidin vs.&#160;neutravidin agarose to isolate the biotinylated protein complexes should be determined on case-by-case basis. In our experience streptavidin agarose provides efficient isolation of the biotinylated protein complexes with minimal binding of nonbiotinylated proteins, unlike the neutravidin agarose where the nonspecific binding occurs at levels that interfere with the assay results (unpublished observation).

All living cells process information by trafficking cargo, such as extracellular ligands, microorganisms, nutrients, transmembrane proteins and lipids from the plasma membrane to endocytic vesicles (i.e. endocytosis). A reciprocal process called recycling balances endocytosis and returns much of the internalized membrane and cargo to the cell surface.&#160; The balance between endocytosis and recycling controls the plasma membrane composition and provides cells with information that has been resolved in time and space. Endocytosis and recycling are master regulators of diverse cellular functions such as nutrient uptake and metabolism, development, proliferation, differentiation and polarity, reprogramming, migration, cell adhesion and migration, cytokinesis, and neurotransmission1-3. Endocytic and recycling pathways are very dynamic and highly coordinated and allow cells to turn over the equivalent of the entire&#160;plasma membrane 1-5x&#160;per hour.
The cell-based L-glutahione protection assays are useful to study endocytosis and recycling of transmembrane proteins including receptors, channels, transporters, and adhesion molecules in epithelial and nonepithelial cells4-8. We have previously studied endocytosis and recycling of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in&#160;human airway epithelial cells and HEK293 cells9-15. The biotinylation-based assays described in the manuscript are optimized for examining endocytosis and recycling in epithelial cells cultured under polarizing conditions on semipermeable growth supports. These protocols can be modified to study endocytosis and recycling of proteins in epithelial cells cultured in plastic tissue culture dishes or in nonepithelial cells. Figures 1&#160;and 2 contain examples of endocytic and recycling assays in epithelial and nonepithelial cells.
Endocytic assays are performed as previously described9-15. Cells are cultured on collagen coated semipermeable growth supports11,14. Alternatively, cells can be cultured in collagen coated plastic tissue culture dishes10,15. Cells are cooled rapidly to 4 &#186;C to stop membrane trafficking and the plasma membrane proteins are labeled at 4 &#176;C with a cell membrane impermeable biotin. Biotin reacts with &#949;-amine of lysine residues and the disulfide bond is thiol-cleavable. After biotinylation cells are incubated at 37 &#176;C to induce protein trafficking and load endocytic vesicles for 2.5, 5.0, 7.5, or 10 min. Subsequently, cells are cooled to 4 &#176;C and the disulfide bond in biotin covalently attached to plasma membrane proteins is reduced with L-glutathione (GSH). At this point in the protocol, only proteins that were endocytosed from the plasma membrane are protected from GSH and thus, remain biotinylated. Cells remaining at 4 &#176;C after biotinylation without incubation at 37 &#186;C or the GSH treatment would serve to determine the amount of CFTR biotinylated at time zero. Cells remaining at 4 &#176;C after biotinylation without incubation at 37 &#186;C but with GSH treatment would serve to determine efficiency of the disulfide bonds reduction. Following the above described treatments, cells are lysed, biotinylated proteins are isolated by streptavidin agarose, eluted into SDS sample buffer, and separated by SDS-PAGE. The protein of interest is detected in the biotinylated samples by western blotting. The amount of biotinylated protein at 4 &#186;C at time zero (without the 37 &#186;C warming) is considered 100%. The amount of protein remaining biotinylated after GSH treatment at 4 &#186;C is considered background and is subtracted from the amount of protein remaining biotinylated after warming to 37 &#186;C at different time points. Protein endocytosis is calculated after subtracting the background and is expressed as the percent of biotinylated protein at each time point after warming to 37 &#186;C compared to the amount of biotinylated protein present at time zero.
Recycling assays are performed as previously described11,16. Cells are cultured on collagen coated semipermeable growth supports11. Alternatively, cells can be cultured on collagen coated plastic tissue culture dishes13. Cells are warmed to 37 &#176;C after biotinylation to load endocytic vesicles with biotinylated proteins. The time of first incubation at 37 &#186;C is determined by the time when endocytosis of the protein of interest reaches maximum during the linear increase of the endocytic signal. The time is protein specific and may depend on the cell type and cell culture conditions. In our experience CFTR endocytosis reached maximum at 5.0 or 7.5 min13,14 (Figures&#160;1 and 2). Subsequently, cells are cooled immediately to 4 &#176;C and the disulfide bond in biotin attached to plasma membrane&#160;proteins is reduced with GSH. Next, cells are either lysed to determine the amount of endocytosed protein of interest or warmed again to 37 &#176;C for different periods of time to allow endocytosed biotinylated protein of interest to recycle to the plasma membrane. Cells are then cooled again to 4 &#176;C, and the disulfide bond on biotin attached to proteins recycled to the plasma membranes is reduced with GSH. Recycling of the protein of interest is determined from the difference between the amount of biotinylated protein after the first and second GSH treatment.
The feasibility of the endocytic and recycling assays depends on several factors. First, formation of cell monolayers is a prerequisite and cells that do not form monolayer or grow as multilayers are not suitable for assays described in this manuscript. Second, the abundance of the protein of interest at the cell surface and presence of an antibody to detect the protein by western blotting are critical. We recommend that the steady state abundance of the protein is first determined in whole cell lysates (WCL). Third, the ability to biotinylate the specific cell surface protein should be tested. Biotin attaches to lysine residues. Thus, the efficiency of biotinylation depends in part on the number of lysine residues in the protein&#8217;s extracellular domain. Accordingly, we recommend screening the protein sequence to determine whether lysine residues are present in the extracellular domain(s). Not all extracellular domain lysine residues may be equally accessible to biotin due to protein folding. Hence, protein biotinylation at steady state followed by western blotting should be performed to determine not only the steady state abundance of the protein at the cell surface but also to examine feasibility of the biotinylation-based assays for the protein of interest.
This protocol is optimized for examining endocytosis and recycling of wild type CFTR in human airway epithelial cells CFBE41o- cultured on 24 mm semipermeable growth supports in air-liquid interface9,10,13-15. CFTR polarizes to the apical membrane domain; thus, the protocol describes biotinylation of the apical membrane domain. Biotinylation of the basolateral membrane domain will be required to study endocytosis and recycling of proteins polarizing to the basolateral membrane. The endocytic assay protocol described in this manuscript has 6 conditions: Biotinylated only (BT = time zero; sample a); GSH control (GSH; sample b); and the 2.5, 5.0, 7.5, or 10 min endocytic time points (samples c; Table 1). The number and/or length of endocytic time points in the protocol can be modified as needed.
The recycling assay is performed after determining the time point when endocytosis of the protein of interest reaches maximum during the linear increase of the endocytic signal. This time point will be used to load endocytic vesicles with the protein of interest prior to inducing recycling. The time is protein dependent and may differ between cell types and culture conditions15. We have previously established that CFTR endocytosis reached plateau at the 7.5&#160;min time point in human airway epithelial cells CFBE41o- stably expressing CFTR15. By contrast, CFTR endocytosis reached plateau at the 5.0&#160;min time point in HEK293 cells stably expressing CFTR13. The recycling assay protocol described in this manuscript has 5 conditions: Biotinylated only (BT = time zero; sample a); GSH control (GSH; sample b); 5.0 min endocytosis (Endo; sample c), 5.0 min endocytosis followed by the 2.5 or 5.0&#160;min recycling time points (Rec; samples d; Table 2). The number and/or length of recycling time points in the protocol can be modified as needed.

<table border="0" cellpadding="0" cellspacing="0" width="682">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CFBE41o- cells</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">reference10,11,13,14,15</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEK293 cells</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td>reference13</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Minimal Essential Medium (MEM)</td>
			<td style="width:180px;">Gibco</td>
			<td style="width:119px;">11095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-Glutamine</td>
			<td style="width:180px;">Cellgro</td>
			<td style="width:119px;">25-005-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin</td>
			<td rowspan="2" style="width:180px;">Sigma</td>
			<td rowspan="2" style="width:119px;">P0781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptomycin</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Puromycin</td>
			<td style="width:180px;">InvivoGen</td>
			<td style="width:119px;">ant-pr-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PureCol&#160;Bovine Collagen&#160;Solution&#160;Type I</td>
			<td rowspan="4" style="width:180px;">Advanced Biomatrix&#160;</td>
			<td rowspan="4" style="width:119px;">5005-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PureCo&#160;Bovine Collagen&#160;Solution&#160;Type I</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td rowspan="2" style="width:180px;"></td>
			<td rowspan="2" style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Culture Reagents</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Dulbecco&#8217;s Phosphate Buffered Saline (PBS)</td>
			<td style="width:180px;">Gibco</td>
			<td style="width:119px;">14190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium chloride</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:119px;">C3306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium chloride</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:119px;">M2670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:119px;">S3014</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">EZLink Sulfo-NHS-SS-Biotin</td>
			<td style="width:180px;">Pierce</td>
			<td style="width:119px;">21331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-Glutathione reduced (GSH)</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:119px;">G6529</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptavidin-agarose resin</td>
			<td style="width:180px;">Thermo Fisher</td>
			<td style="width:119px;">20353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium hydroxide</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:119px;">224165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal bovine serum&#160;</td>
			<td style="width:180px;">Gemini Bioscience</td>
			<td style="width:119px;">100-106</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Milli-Q water</td>
			<td style="width:180px;">Millipore</td>
			<td style="width:119px;">Advantage System</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:180px;">Bio Rad</td>
			<td style="width:119px;">161-0407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:180px;">FisherScientific</td>
			<td style="width:119px;">G33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laemli Sample buffer</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:119px;">161-0737</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dithiothreitol (DTT)</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:119px;">646563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Complete Protease&#160;Inhibitor cocktail</td>
			<td rowspan="2" style="width:180px;">Roche</td>
			<td rowspan="2" style="width:119px;">1697498</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Complete Protease&#160;Inhibitor cocktail</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptavidin beads</td>
			<td style="width:180px;">Thermo Scientific</td>
			<td style="width:119px;">20353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chemiluminescence</td>
			<td rowspan="2" style="width:180px;">Perkin Elmer</td>
			<td rowspan="2" style="width:119px;">NEL104001EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reagents</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nonfat dry milk</td>
			<td style="width:180px;">Carnation</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween-20</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:119px;">170-6539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris-buffered Saline (TBS)</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:119px;">170-6435</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:180px;">Gibco&#160;</td>
			<td style="width:119px;">15120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primary and secondary&#160;</td>
			<td style="width:180px;"></td>
			<td rowspan="7" style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Antibodies for Western Blotting</td>
			<td style="width:180px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Mouse monoclonal Anti-CFTR 596</td>
			<td style="width:180px;">Cystic Fibrosis Foundation Therapeutics, Inc., Chapel Hill, NC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:180px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;"></td>
			<td style="width:180px;">BD Biosciences, San Jose, CA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mouse monoclonal&#160;</td>
			<td style="width:180px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-ezrin&#160;</td>
			<td style="width:180px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Running buffer 10x</td>
			<td style="width:180px;">BioRad</td>
			<td style="width:119px;">161-0772</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Transfer buffer 10x&#160;(without SDS)</td>
			<td rowspan="2" style="width:180px;">BioRad</td>
			<td rowspan="2" style="width:119px;">161-0771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">(without SDS)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:180px;">Fisher</td>
			<td style="width:119px;">A4129</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Coulter counter</td>
			<td style="width:180px;">Beckman Coulter</td>
			<td style="width:119px;">Z1Coulter Particle Counter</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Standard Cell&#160;Culture equipment</td>
			<td rowspan="2" style="width:180px;"></td>
			<td rowspan="2" style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Culture equipment</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Cell culture hood with UV light</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stock flasks</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Transwell filters-24mm</td>
			<td style="width:180px;">Corning</td>
			<td style="width:119px;">3412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4 cm cell culture dishes</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Standard cell&#160;culture equipment&#160;</td>
			<td rowspan="2" style="width:180px;"></td>
			<td rowspan="2" style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">culture equipment&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pH meter</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">15 ml and 50 ml conical tubes</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 ml Eppendorf tubes</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rubber policeman</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cold room</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ice buckets and ice</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">27 G needles</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">37 &#186;C Incubator</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thermometer</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benchtop centrifuge</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cooled centrifuge</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrophoresis equipment</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Developer</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Film</td>
			<td style="width:180px;">GE Amersham</td>
			<td style="width:119px;">28-9068-36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PVDF membrane</td>
			<td style="width:180px;">Millipore</td>
			<td style="width:119px;">IPVH00010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ImageJ software</td>
			<td style="width:180px;">NIH</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

the cell-based l-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

CFTR endocytosis was studied in CFBE41o- cells cultured on collagen-coated filters (Figure&#160;1). Biotinylated CFTR was visualized by western blotting with mouse monoclonal antibody, clone 596 and an anti-mouse horseradish peroxidase antibody using the western blotting detection system followed by chemiluminesence. Quantification of biotinylated CFTR was performed by densitometry using exposures within the linear dynamic range of the film. CFTR endocytosis was calculated after subtracting the background and was expressed as the percent of biotinylated CFTR at each time point after warming to 37 &#186;C compared to the amount of biotinylated CFTR present at time zero (Figures &#160;1A and&#160;1B). CFTR endocytosis was linear between 0-7.5 min. Experiments in which the background CFTR was &#62;10% were excluded due to inefficient GSH treatment (Figure&#160;1D).
CFTR recycling was studied in HEK293 cells cultured in collagen-coated tissue culture dishes (Figure&#160;2). CFTR endocytosis was linear between 0.0-5.0 min and reached maximum at the 5.0&#160;min time point (Figure 2A), thus cells were incubated at 37 &#186;C for 5.0 min to load endocytic vesicles with biotinylated proteins including CFTR (Figures 2B and&#160;2C). Recycling of endocytosed CFTR was calculated as the difference between the amount of biotinylated CFTR after the first and second GSH treatment.
Table 1. Endocytic assays.
<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td valign="top" width="100">Endocytosis</td>
			<td>BT</td>
			<td>GSH</td>
			<td>Endo-2.5</td>
			<td>Endo-5.0</td>
			<td>Endo-7.5</td>
			<td>Endo-10.0</td>
		</tr>
		<tr>
			<td valign="top" width="137">Sample</td>
			<td valign="top" width="91">a</td>
			<td valign="top" width="81">b</td>
			<td valign="top" width="113">c2.5 </td>
			<td valign="top" width="105">c5.0</td>
			<td valign="top" width="120">c7.5</td>
			<td valign="top" width="143">c10.0</td>
		</tr>
		<tr>
			<td valign="top" width="137">Biotin</td>
			<td valign="top" width="91">+</td>
			<td valign="top" width="81">+</td>
			<td valign="top" width="113">+</td>
			<td valign="top" width="105">+</td>
			<td valign="top" width="120">+</td>
			<td valign="top" width="143">+</td>
		</tr>
		<tr>
			<td valign="top" width="137">37 &#186;C</td>
			<td valign="top" width="91">(-)</td>
			<td valign="top" width="81">(-)</td>
			<td valign="top" width="113">2.5 min</td>
			<td valign="top" width="105">5.0 min</td>
			<td valign="top" width="120">7.5 min</td>
			<td valign="top" width="143">10 min</td>
		</tr>
		<tr>
			<td valign="top" width="137">GSH</td>
			<td valign="top" width="91">(-)</td>
			<td valign="top" width="81">+</td>
			<td valign="top" width="113">+</td>
			<td valign="top" width="105">+</td>
			<td valign="top" width="120">+</td>
			<td valign="top" width="143">+</td>
		</tr>
	</tbody>
</table>
Table 2. Recycling assay.
<table border="1" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Recycling</td>
			<td>BT</td>
			<td>GSH</td>
			<td>Endo-5</td>
			<td>Rec-2.5</td>
			<td>Rec-5.0</td>
		</tr>
		<tr>
			<td valign="top" width="137">Sample</td>
			<td valign="top" width="90">a</td>
			<td valign="top" width="83">b</td>
			<td valign="top" width="90">c</td>
			<td valign="top" width="105">d2.5</td>
			<td valign="top" width="105">d5.0</td>
		</tr>
		<tr>
			<td valign="top" width="137">Biotin</td>
			<td valign="top" width="90">+</td>
			<td valign="top" width="83">+</td>
			<td valign="top" width="90">+</td>
			<td valign="top" width="105">+</td>
			<td valign="top" width="105">+</td>
		</tr>
		<tr>
			<td valign="top" width="137">1st 37 &#186;C</td>
			<td valign="top" width="90">(-)</td>
			<td valign="top" width="83">(-)</td>
			<td valign="top" width="90">5 min</td>
			<td valign="top" width="105">5 min</td>
			<td valign="top" width="105">5 min</td>
		</tr>
		<tr>
			<td valign="top" width="137">1st GSH</td>
			<td valign="top" width="90">(-)</td>
			<td valign="top" width="83">(-)</td>
			<td valign="top" width="90">+</td>
			<td valign="top" width="105">+</td>
			<td valign="top" width="105">+</td>
		</tr>
		<tr>
			<td valign="top" width="137">2nd 37 &#186;C</td>
			<td valign="top" width="90">(-)</td>
			<td valign="top" width="83">(-)</td>
			<td valign="top" width="90">+</td>
			<td valign="top" width="105">+</td>
			<td valign="top" width="105">+</td>
		</tr>
		<tr>
			<td valign="top" width="137">2nd GSH</td>
			<td valign="top" width="90">(-)</td>
			<td valign="top" width="83">(-)</td>
			<td valign="top" width="90">(-)</td>
			<td valign="top" width="105">2.5 min</td>
			<td valign="top" width="105">5 min</td>
		</tr>
	</tbody>
</table>
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50867/50867fig1highres.jpg" src="/files/ftp_upload/50867/50867fig1.jpg" /> 
Figure 1. Summary of endocytic assays performed to determine CFTR endocytosis in CFBE41o- cells. Cells were cultured on collagen-coated filters. Representative western blots (A), representative densitometry values (B) and summary of experiments (C) demonstrating CFTR endocytosis as a function of time. Selective cell surface biotinylation and western blotting were used to determine the abundance of plasma membrane CFTR. Protein abundance was quantified by densitometry using exposures within the linear dynamic range of the film. At time zero, the amount of biotinylated (BT) CFTR was considered 100% (Table 1: sample a). At time zero, the amount of BT CFTR remaining after GSH treatment was considered a CFTR background (sample b; please, note this is a different background than the one subtracted from all samples as shown in Figure&#160;1B). Background CFTR was 6.7 &#177; 0.9% (mean &#177; S.E.M.) in the experiments included for analysis. Background CFTR was subtracted from the BT CFTR after the 2.5, 5.0, 7.5, or 10&#160;min warming at 37 &#186;C (samples c minus sample b). CFTR endocytosis was expressed as the percent of CFTR remaining biotinylated at the 2.5, 5.0, 7.5, or 10 min&#160;time points after subtracting background CFTR. CFTR endocytosis was linear between zero and 7.5 min. Ezrin abundance in the whole cell lysate (WCL) was used as a loading control. 4 experiments/group. Experiments in which the background CFTR was &#62;10% were excluded due to inefficient GSH treatment (D). The amount of biotinylated CFTR in the GSH control (sample b) in the excluded experiment was 14.5%.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50867/50867fig2highres.jpg" src="/files/ftp_upload/50867/50867fig2.jpg" /> 
Figure 2. Summary of endocytic and recycling assays performed in HEK293 cells stably expressing CFTR. Cells were cultured in collagen-coated tissue culture plates. Summary of data demonstrating that CFTR endocytosis was linear between 0-5 min (A). Thus, in the recycling assays endocytic vesicles were loaded with biotinylated (BT) proteins including CFTR by warming at 37 &#186;C for 5 min. Protein abundance was quantified by densitometry using exposures within the linear dynamic range of the film. Representative western blot (B), representative densitometry values (C), and summary of experiments (D) demonstrating CFTR recycling as a function of time. At time zero, the amount of BT&#160;CFTR was considered 100% (Table 2: sample a). At time zero, the amount of BT CFTR remaining after GSH treatment was considered a CFTR background (sample b; please, note this is a different background than the one subtracted from all samples as shown in C). Experiments in which the background CFTR was &#62;10% were excluded due to inefficient GSH treatment. Endocytic vesicles were loaded with BT proteins including CFTR by incubation at 37 &#186;C for 5 min followed by the GSH treatment to cleave biotin from proteins remaining at the plasma membrane (samples c and d). The amount of BT CFTR after the 5&#160;min warming at 37 &#186;C followed by the GSH treatment represents endocytosed CFTR (sample c). Following the 5&#160;min warming at 37 &#186;C and the first GSH treatment cells were warmed again at 37 &#186;C for 2.5 or 5.0 min to allow the endocytosed proteins to recycle to the plasma membrane and the biotin on recycled CFTR was reduced by the second GSH treatment (samples d). At this point only the CFTR that has not recycled from endosomes to the plasma membrane remained biotinylated (samples d). CFTR recycling was calculated as the difference between BT CFTR after the first GSH treatment (sample c) and second GSH treatment at 2.5 and 5.0 min (samples d) and was expressed as the percent of endocytosed CFTR. CFTR recycling was rapid and reached maximum by 2.5 min. Ezrin abundance in the whole cell lysate (WCL) was used as a loading control. 3 experiments/group.

Watch this Scientific Journal Video about The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins at JoVE.com

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

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10.4K Views.  Children's Hospital of Pittsburgh of UPMC. Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Video: The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1&#160;was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS.
First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a &#34;slope of slopes&#34; plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example.

This paper provides a step by step guide to the fluctuation analysis technique k-Space Image Correlation Spectroscopy (kICS) for measuring diffusion coefficients of fluorescently labeled plasma membrane proteins in live mammalian cells.

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...