This project was funded by NIH Grants 1R01AI125640-01 and the Rheumatology Research Foundation.

1. Transfection of Cells
<ol>
	<li>Seed HEK 293T cells into twelve 10 cm plates (5 x 106 cells per plate) the day before transfection (Figure 1). Perform the cell counting using a hemocytometer. For each plate, use 10 mL of DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Incubate the cells at 37 &#176;C in 5% CO2.</li>
	<li>Once the cells are 80-90% confluent, transfect 5 HEK 293T plates using a lipid-based transfection reagent with one of the following plasmids: a SHP2-expressing vector, a PD-1-GFP-expressing vector [a wild type (WT) version of PD-1], an ITIM-mutated version (Y223F) of a PD-1-GFP-expressing vector, and an ITSM-mutated version (Y248F) of a PD-1-GFP-expressing vector (Figure 1). 
	NOTE: Two plates will be transfected with the WT PD-1-GFP-expressing vector. One additional plate will serve as a non-transfected cells control (Figure 1).</li>
	<li>Perform the transfection as per the manufacturer&#39;s protocol for adherent cells in 10 cm plates. 
	NOTE: In the mutated versions of PD-1, the tyrosine (Y) in each motif was replaced by phenylalanine (F) that cannot be phosphorylated.</li>
	<li>Transfect a second identical set of five plates and one additional plate serving as a non-transfected control for the measurement of expressed protein amount [input; also known as whole cell lysate (WCL)] before the immunoprecipitation (Figure 1).</li>
	<li>Incubate the transfected cells for 48 h in 37 &#176;C, 5% CO2 in a tissue culture incubator. 
	NOTE: To ensure that transfection has successfully occurred, examine the cells for GFP expression using a fluorescent microscope.</li>
</ol>
2. Promoting Phosphorylation in Transfected Cells
<ol>
	<li>Following incubation, prepare fresh pervanadate by mixing 50 &#181;L of sodium-orthovanadate (from 100 mM stock) with 50 &#181;L of 30% H2O2. 
	NOTE: The mixture should change into a yellowish color. Pervanadate is a cell-permeable phosphatase inhibitor that promotes phosphorylation of tyrosine residues13. In this assay, it is used to robustly induce phosphorylation of the tails of PD-1.</li>
	<li>To phosphorylate the tagged versions of PD-1, remove the media from the PD-1-GFP-transfected cells and add 10 mL of plain DMEM (without serum or antibiotics) together with 10 &#181;L of pervanadate (step 2.1) to each 10 cm plate (resulting in eight plates expressing different versions of PD-1) (Figure 1). Place the plates at room temperature in the dark for 15 min. 
	NOTE: The SHP2-transfected cells (two plates; Figure 1) and the two non-transfected control plates are not treated with pervanadate, since these plates will serve as the source for the active SHP2 enzyme.</li>
	<li>Wash the cells with 5 mL of ice-cold 1x PBS. Repeat this step twice.</li>
</ol>
3. Immunoprecipitation
NOTE: The following steps should be performed on ice or at 4 &#176;C.
<ol>
	<li>Supplement the lysis buffer (50 mM Tris-HCl at pH 7.2, 250 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10% glycerol) with protease inhibitors (dissolve 1 tablet in 10 mL of lysis buffer) and with 1 mM sodium orthovanadate. Add 500 &#181;L of ice-cold lysis buffer to the cells and remove and collect the cells from the plates immediately using a cell scraper. 
	NOTE: It is important to add the sodium orthovanadate only to the lysis buffer that will be used for the PD-1-GFP-transfected plates, since the SHP2-transfected plates and non-transfected control plates must retain phosphatase activity.</li>
	<li>Transfer the lysates into 1.5 mL cold tubes and rotate them at 0.005 x g, 4 &#176;C for 30 min.</li>
	<li>To collect the post nuclei supernatant (PNS) from the lysates, spin down the lysates for 10 min at 10,000 x g at 4 &#176;C and transfer the supernatants into new tubes. Discard the pellet. Store the supernatants of the second set of six plates (Figure 1) on ice for later WCL analysis.</li>
	<li>Preparation of the anti-GFP beads for immunoprecipitation of PD-1-GFP from the PD-1-GFP-transfected lysates of the first set (four plates) (Figure 1).
	<ol>
		<li>To prevent settling of the beads, gently shake the bottle with the beads before opening. Remove 40 &#181;L of the anti-GFP beads from the slurry per each condition.</li>
		<li>Spin at 500 x g for 3 min at 4 &#176;C and remove the supernatant to wash the beads. 
		NOTE: It is important to minimize the contact between the pipette plastic tips and agarose beads to prevent loss. It is recommended to cut the edge of a 200 &#181;L tip before transferring the beads to another tube.</li>
		<li>Resuspend the beads in 80 &#181;L of lysis buffer (per sample).</li>
	</ol>
	</li>
	<li>Add the washed beads directly to the cell lysate (PNS) from the PD-1-GFP-expressing cells of the first set (four plates) (step 3.3). Rotate at 0.005 x g for 30 min at 4 &#176;C to immunoprecipitate the GFP-tagged proteins.</li>
	<li>Wash the beads with 1 mL of cold lysis buffer (without orthovanadate) 3 times. Centrifuge the tube at 2,500 x g for 10 s. 
	NOTE: When adding the lysis buffer to the beads, add it directly onto the beads without touching them with the tips. There is no need to pipette up and down during the washes.</li>
	<li>Equally divide the lysate of the active SHP2 from the first set (one plate; step 3.3) and add it to three tubes of the washed PD-1-GFP-containing beads (the WT PD-1-GFP and the two different phosphdeficient mutations, Y223F and Y248F). Add one-third of the volume from the lysate of the non-transfected cells to the second tube of the WT PD-1-GFP beads. Discard the remaining two-thirds.</li>
	<li>Incubate the beads for 4 h at 4 &#176;C with gentle rotation (0.005 x g).</li>
	<li>Wash the beads twice with 1 mL of cold lysis buffer (without pervanadate or orthovanadate), as reported in step 3.6.</li>
	<li>Add 80 &#181;L of lysis buffer (without pervanadate or orthovanadate) per sample ensuring that the total volume in each tube is 100 &#181;L (20 &#181;L of beads and 80 &#181;L of lysis buffer). Mix gently and transfer 50 &#181;L from each tube into two fresh 1.5 mL tubes. 
	NOTE: Following this step, there will be two tubes filled with a mixture that contains beads and lysis buffer: one tube will be used for testing the co-immunoprecipitated SHP2 by western blotting, and the second will be used for the phosphatase activity assay.</li>
</ol>
4. Phosphatase Activity Assay
<ol>
	<li>Wash the beads once with phosphatase wash buffer (30 mM Hepes at pH 7.4 and 120 mM NaCl). Remove the supernatant completely.</li>
	<li>Add 100 &#181;L of assay buffer (30 mM Hepes at pH 7.4, 120 mM NaCl, 5 mM DTT, 10 mM p-nitrophenylphosphate) to the beads, and incubate at 30 &#176;C for 30 min under gentle agitation. NOTE: Incubation time may vary, but wait until the buffer turns yellow upon dephosphorylation.</li>
	<li>To terminate the reaction, add 50 &#181;L of 1 M NaOH when the buffer turns yellow.</li>
	<li>Spin down at 2,500 x g for 10 s and transfer 50 &#181;L of the supernatant to two wells (duplicates with 50 &#181;L per well) of half-area in a 96-well plate. Read the absorbance at 405 nm.</li>
	<li>Express the results as relative optical density (OD) over the control wild-type version of PD-1-GFP.</li>
</ol>
5. SHP2 Western Blot Analysis
<ol>
	<li>Spin down the beads and remove the supernatant.</li>
	<li>Add 20 &#181;L of 2x Laemmli buffer (see Materials Table) to the beads and boil at 95 &#176;C for 5 min.</li>
	<li>Measure the protein concentration of the input controls (second set) using a BCA kit (see Materials Table). Transfer 30 &#181;L of the most diluted sample to a new tube. Dilute the rest of the input controls with lysis buffer to the same concentration as the most diluted sample, and transfer 30 &#181;L from each of them to a new tube. 
	NOTE: Following this step, there will be 4 tubes with 30 &#181;L each, at similar protein concentrations, representing the input controls lysates.</li>
	<li>Add equal volumes of 2x Laemmli buffer to the lysates and boil at 95 &#176;C for 5 min. Spin down the beads and load the supernatant on 4&#8211;20% SDS-PAGE Tris-based gel (see Materials Table) for western blot analysis. In addition, load 50 &#181;L from each sample of the second set.</li>
	<li>Continue western blot analysis according to the protocol previously described12.</li>
</ol>

<ol>
	<li>Montor, W. R., Salas, A., Melo, F. H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+tyrosine+kinases+and+downstream+pathways+as+druggable+targets+for+cancer+treatment:+the+current+arsenal+of+inhibitors.">Receptor tyrosine kinases and downstream pathways as druggable targets for cancer treatment: the current arsenal of inhibitors.</a> Molecular Cancer. 17 (1), (2018).</li><li>Ngoenkam, J., Schamel, W. W., Pongcharoen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selected+signalling+proteins+recruited+to+the+T-cell+receptor-CD3+complex.">Selected signalling proteins recruited to the T-cell receptor-CD3 complex.</a> Immunology. 153 (1), 42-50 (2018).</li><li>Azoulay-Alfaguter, I., Strazza, M., Pedoeem, A., Mor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+coreceptor+programmed+death+1+inhibits+T-cell+adhesion+by+regulating+Rap1.">The coreceptor programmed death 1 inhibits T-cell adhesion by regulating Rap1.</a> Journal of Allergy and Clinical Immunology. 135 (2), 564-567 (2015).</li><li>Chemnitz, J. M., Parry, R. V., Nichols, K. E., June, C. H., Riley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHP-1+and+SHP-2+associate+with+immunoreceptor+tyrosine-based+switch+motif+of+programmed+death+1+upon+primary+human+T+cell+stimulation,+but+only+receptor+ligation+prevents+T+cell+activation.">SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation.</a> The Journal of Immunology. 173 (2), 945-954 (2004).</li><li>Patsoukis, N., 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=Selective+effects+of+PD-1+on+Akt+and+Ras+pathways+regulate+molecular+components+of+the+cell+cycle+and+inhibit+T+cell+proliferation.">Selective effects of PD-1 on Akt and Ras pathways regulate molecular components of the cell cycle and inhibit T cell proliferation.</a> Science Signaling. 5 (230), 46 (2012).</li><li>Pentcheva-Hoang, T., Chen, L., Pardoll, D. M., Allison, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+death-1+concentration+at+the+immunological+synapse+is+determined+by+ligand+affinity+and+availability.">Programmed death-1 concentration at the immunological synapse is determined by ligand affinity and availability.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (45), (2007).</li><li>Yokosuka, T., 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=Programmed+cell+death+1+forms+negative+costimulatory+microclusters+that+directly+inhibit+T+cell+receptor+signaling+by+recruiting+phosphatase+SHP2.">Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2.</a> The Journal of Experimental Medicine. 209 (6), 1201-1217 (2012).</li><li>Hui, E., 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=T+cell+costimulatory+receptor+CD28+is+a+primary+target+for+PD-1-mediated+inhibition.">T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition.</a> Science. 355 (6332), 1428-1433 (2017).</li><li>Sheppard, K. 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=PD-1+inhibits+T-cell+receptor+induced+phosphorylation+of+the+ZAP70/CD3zeta+signalosome+and+downstream+signaling+to+PKCtheta.">PD-1 inhibits T-cell receptor induced phosphorylation of the ZAP70/CD3zeta signalosome and downstream signaling to PKCtheta.</a> FEBS Letters. 574 (1-3), 37-41 (2004).</li><li>Okazaki, T., Maeda, A., Nishimura, H., Kurosaki, T., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-1+immunoreceptor+inhibits+B+cell+receptor-mediated+signaling+by+recruiting+src+homology+2-domain-containing+tyrosine+phosphatase+2+to+phosphotyrosine.">PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine.</a> Proceedings of the National Academy of Sciences of the United States of America. 98 (24), 13866-13871 (2001).</li><li>Sun, J., 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=Antagonism+between+binding+site+affinity+and+conformational+dynamics+tunes+alternative+cis-interactions+within+Shp2.">Antagonism between binding site affinity and conformational dynamics tunes alternative cis-interactions within Shp2.</a> Nature Communications. 4, 2037 (2013).</li><li>Peled, 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=Affinity+purification+mass+spectrometry+analysis+of+PD-1+uncovers+SAP+as+a+new+checkpoint+inhibitor.">Affinity purification mass spectrometry analysis of PD-1 uncovers SAP as a new checkpoint inhibitor.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (3), E468-E477 (2018).</li><li>Jang, S. H., 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=A+protein+tyrosine+phosphatase+inhibitor,+pervanadate,+inhibits+angiotensin+II-Induced+beta-arrestin+cleavage.">A protein tyrosine phosphatase inhibitor, pervanadate, inhibits angiotensin II-Induced beta-arrestin cleavage.</a> Molecules and Cells. 28 (1), 25-30 (2009).</li><li>Pluskey, S., Wandless, T. J., Walsh, C. T., Shoelson, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+stimulation+of+SH-PTP2+phosphatase+activity+by+simultaneous+occupancy+of+both+SH2+domains.">Potent stimulation of SH-PTP2 phosphatase activity by simultaneous occupancy of both SH2 domains.</a> The Journal of Biological Chemistry. 270 (7), 2897-2900 (1995).</li><li>McAvoy, T., Nairn, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serine/threonine+protein+phosphatase+assays.">Serine/threonine protein phosphatase assays.</a> Current Protocols in Molecular Biology. , (2010).</li><li>Shlapatska, L. 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=CD150+association+with+either+the+SH2-containing+inositol+phosphatase+or+the+SH2-containing+protein+tyrosine+phosphatase+is+regulated+by+the+adaptor+protein+SH2D1A.">CD150 association with either the SH2-containing inositol phosphatase or the SH2-containing protein tyrosine phosphatase is regulated by the adaptor protein SH2D1A.</a> The Journal of Immunology. 166 (9), 5480-5487 (2001).</li></ol>

The authors have nothing to disclose.

Receptor-enzyme interactions are crucial for intracellular signaling. Many enzymes are recruited to receptors through SH2 domains binding to phosphorylated tyrosines that decorate tails of the same receptors. However, enzymes are often folded into closed inactive conformations, and activation requires a conformational change11 that can be mediated by other domains of the same receptor. The assay described here measures the interactions between receptors and enzymes as well as the activity induced ...

Catalytic receptors and receptor tyrosine kinases are transmembrane proteins in which binding of an extracellular ligand causes enzymatic activity on the intracellular side1. Some receptors possess both receptor and enzymatic functions, while others recruit specific enzymes such as kinases and phosphatases to their cytoplasmic tails. Recruitment of an enzyme to the receptor&#39;s tail and the subsequent catalytic action of this enzyme are two separate processes that are not always regulated by the same protein domains2. Unfortunately, there are no specific tools to assess both the interaction and enzymatic activity simultaneously. The functional co-immunoprecipitation assay described here is a useful method to dissect the recruitment of an enzyme to the tail of a receptor from its activation. This assay utilizes immunoprecipitation of tagged receptors by antibody-coated beads. Subsequently, both an enzymatic activity assay and western blot analysis on beads are performed. The overall goal of this method is to uncover which protein domains are necessary for interactions between receptors and enzymes (assessed by western blot analysis) and which domains are obligatory for complete activation of the enzymes (measured by on-bead enzymatic activity assay). It is significant to develop tools for studying the separate functions of receptor-associated enzymes due to their involvement in the pathogenesis of human diseases. Moreover, further understanding the mechanisms of action of these proteins may help the design of novel therapeutic interventions.
Programmed death-1 (PD-1) is an inhibitory receptor on the surface of T cells and is required for limiting excessive T cell responses. In recent years, anti-PD-1 antibodies have been implicated in the treatment of multiple malignancies1,2. PD-1 ligation restrains numerous T cell functions, including proliferation, adhesion, and secretion of multiple cytokines3,4,5. PD-1 is localized to the immunological synapse, the interface between T cells and antigen-presenting cells6, where it colocalizes with the T cell-receptor (TCR)7. Subsequently, the tyrosine phosphatase SHP2 [Src homology 2 (SH2) domain containing tyrosine phosphatase 2] is recruited to the cytoplasmic tail of PD-1, leading to dephosphorylation of key tyrosine residues within the TCR complex and its associated proximal signaling molecules3,4,5,8,9. The cytoplasmic tail of PD-1 contains two tyrosine motifs, an immunoreceptor tyrosine-based inhibitory motif (ITIM), and an immunoreceptor tyrosine based-switch motif (ITSM)10. Both motifs are phosphorylated upon PD-1 ligation9,10. Mutagenesis studies have revealed a primary role for the ITSM in SHP2 recruitment, as opposed to the ITIM, whose role in PD-1 signaling and function is not clear4.
SHP2 adopts either a closed (folded), inhibited conformation or an open (extended), active conformation11. The contribution of each tail domain of PD-1 to SHP2 binding or activation has not yet been elucidated. To answer this question, we developed an assay that enables parallel testing of the recruitment of SHP2 to the tail of PD-1 and its activity12. We employed co-immunoprecipitation and an on-bead phosphatase activity assay to test both the interaction and enzymatic activity in parallel. Using this assay, we show that the ITSM of PD-1 is sufficient to recruit SHP2 to the tail of PD-1, while the ITIM of PD-1 is needed to fully extend and activate the enzyme.
There are many receptors that have several adjacent domains in their cytoplasmic tails. The functional co-immunoprecipitation assay can uncover the role of specific domains that are necessary for either protein recruitment or enzymatic activation.

<table>
	<tbody>
		<tr>
			<td>PBS</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td>Phosphate Buffered Saline</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5424-R</td>
			<td>1.5 ml</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%) Phenol Red</td>
			<td>Gibco</td>
			<td>25200114</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated FBS</td>
			<td>Denville</td>
			<td>FB5001-H</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>Penicillin / Streptomycin</td>
			<td>Fisher</td>
			<td>BP295950</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose without L-glutamine</td>
			<td>Lonza</td>
			<td>BE12-614F</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFect Transfection Reagent</td>
			<td>Qiagen</td>
			<td>301305</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-SHP2</td>
			<td>Santa Cruz</td>
			<td>SC-280</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti&#8211;GFP-agarose</td>
			<td>MBL</td>
			<td>D153-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP</td>
			<td>Roche</td>
			<td>118144600</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Actin</td>
			<td>Santa Cruz</td>
			<td>SC-1616</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK-293 cells</td>
			<td>ATCC</td>
			<td>CRL-1573</td>
			<td></td>
		</tr>
		<tr>
			<td>Orthovanadate</td>
			<td>Sigma</td>
			<td>S6508</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma</td>
			<td>216763</td>
			<td>30%</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td>EDTA-free</td>
		</tr>
		<tr>
			<td>Tris-Glycine SDS Sample Buffer (2X)</td>
			<td>Invitrogen</td>
			<td>LC2676</td>
			<td>Modified Laemmli buffer</td>
		</tr>
		<tr>
			<td>4-20% Tris-Glycine Mini Gels</td>
			<td>Invitrogen</td>
			<td>XP04205BOX</td>
			<td>15-well</td>
		</tr>
		<tr>
			<td>Nitrocellulose membranes</td>
			<td>General Electric</td>
			<td>10600004</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>Sodium chloride</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td>N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Invitrogen</td>
			<td>D1532</td>
			<td>Dithiothreitol</td>
		</tr>
		<tr>
			<td>pNPP</td>
			<td>Sigma</td>
			<td>20-106</td>
			<td>p-Nitrophenyl Phosphate</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S8045</td>
			<td>Sodium hydroxide</td>
		</tr>
		<tr>
			<td>BCA</td>
			<td>Fisher</td>
			<td>23225</td>
			<td>Bi Cinchoninic Acid assay</td>
		</tr>
	</tbody>
</table>

co-immunoprecipitation assay for studying functional interactions between receptors and enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

While the contribution of the ITSM of PD-1 to SHP2 binding is established, the role of the ITIM of PD-1 is less clear. Because SHP2 has two SH2 domains that can bind to two sequential phosphotyrosines on PD-1 (one tyrosine in the ITSM and another in the ITIM), we hypothesized that the ITSM of PD-1 anchors SHP2 to PD-1, while the ITIM of PD-1 facilitates SHP2 activity by stabilizing its open conformational state11,14. To test this,...

Watch this Scientific Journal Video about Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes at JoVE.com

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

co-immunoprecipitation-assay-for-studying-functional-interactions

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Immunology and Infection

14.8K Views.  The Chaim Sheba Medical Center. Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Video: Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure makes the spore extremely stable and resistant and has suggested the use of the spore as a platform to display heterologous molecules. So far, a variety of antigens and enzymes have been displayed on spores of Bacillus subtilis and of a few other species, initially by a recombinant approach and, then, by a simple and efficient nonrecombinant method. The nonrecombinant display system is based on the direct adsorption of heterologous molecules on the spore surface, avoiding the construction of recombinant strains and the release of genetically modified bacteria in the environment. Adsorbed molecules are stabilized and protected by the interaction with spores, which limits the rapid degradation of antigens and the loss of enzyme activity at unfavorable conditions. Once utilized, spore-adsorbed enzymes can be collected easily with a minimal reduction of activity and reused for additional reaction rounds. In this paper is shown how to adsorb model molecules to purified spores of B. subtilis, how to evaluate the efficiency of adsorption, and how to collect used spores to recycle them for new reactions.

This protocol focuses on the use of bacterial spores as a &#34;live&#34; nanobiotechnological tool to adsorb heterologous molecules with various biological activities. The methods to measure the efficiency of adsorption are also shown.

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure ...