The Structural Genomics Consortium is a registered charity (no: 1097737) that receives funds from AbbVie, Bayer AG, Boehringer Ingelheim, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510], Janssen, Merck KGaA (aka EMD in Canada and US), Pfizer, Takeda and the Wellcome Trust [106169/ZZ14/Z].

1. Cell culturing and plating
NOTE: Culture cells with recommended media and test routinely for mycoplasma contamination. HEK293T, MCF7, and C2C12 cells were chosen as examples since these cell lines were successfully used in PRMT assays.
<ol>
	<li>Culture HEK293T, MCF7, and C2C12 in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 U mL&#8722;1), and streptomycin (100 &#181;g mL&#8722;1) in 10 cm tissue-culture treated (TC) dishes.</li>
	<li>For the PRMT8 assay, grow PRMT1 inducible knockdown HEK293T cells in media containing doxycycline (2 &#181;g/mL) for 3 days before assay start.</li>
	<li>To plate the cells, remove and discard media from the plate.</li>
	<li>Add 10 mL of PBS (without Ca+2 and Mg+2 ions) to wash cells and discard the solution.</li>
	<li>Add 1 mL of Trypsin-EDTA (0.25%), incubate for 1 min at room temperature (RT), and then discard the solution. Incubate until cells become round and detach from the plate. Tap the plate to help detach cells, if needed. For hard-to-trypsinize cells, such as C2C12, incubate the plate for 1-2 min at 37 &#176;C. 
	NOTE: Avoid cell exposure to trypsin solution for longer periods (&#62;10 min) as it will reduce cell viability.</li>
	<li>Add 1 mL of prewarmed media to the plate, and gently pipette cells up and down to break-up cell clumps. Transfer cells to a 15 mL tube, and add 3-5 mL of media.</li>
	<li>To measure cell number, mix 10 &#181;L of cells with 10 &#181;L of Trypan blue and transfer 10 &#181;L to hemocytometer or use any other cell counting method.</li>
	<li>Dilute cells to recommended cell density and put 500 &#181;L/well into 24-well TC plates (Table 1). For endogenous assays (PRMT1, PRMT4, PRMT5, PRMT7, and PRMT9), move to Step 3.1.</li>
</ol>
2. Cell transfection
<ol>
	<li>For exogenous assays (PRMT3, PRMT6, PRMT8) transfect HEK293T cells with the recommended amount of DNA (Table 2). HEK293T cells are easy to transfect so any transfection reagent can be used, following the manufacturer&#39;s instruction.</li>
</ol>
3. Compound treatment
NOTE: Do not exceed 0.1% final dimethyl sulfoxide (DMSO) concentration in culture media. Keep the same DMSO concentration in each well. The selective PRMT inhibitors (chemical probes) and their closely related inactive analogs can be found in Table 3.
<ol>
	<li>For endogenous assays (PRMT1, PRMT4, PRMT5, PRMT7, and PRMT9), remove media from cells and replace with 500 &#181;L of media with compound or DMSO alone (control). 
	NOTE: It usually takes 2 days to observe over 80% decrease in R methylation levels.</li>
	<li>For exogenous assays (PRMT3, PRMT6, PRMT8), remove media 4 h after transfection, add 500 &#181;L of media with compound or DMSO alone (control), and incubate for 20-24 h.</li>
</ol>
4. Cell lysate preparation
<ol>
	<li>Remove all media from wells, wash with 100 &#181;L of PBS to remove residual media, and add 60 &#181;L of lysis buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 10 mM MgCl2, 0.5% Triton X-100, 12.5 U mL&#8722;1 benzonase, complete EDTA-free protease inhibitor cocktail) to each well.
	<ol>
		<li>Incubate for less than 1 min at RT, rocking the plate to distribute the lysis buffer over the cells. Then add 3 &#181;L of 20% w/v sodium dodecyl sulfate (SDS), to a final 1 % concentration, and mix by gently shaking. Transfer lysate into microcentrifuge tubes and keep it on ice. 
		NOTE: Add benzonase and protein inhibitor cocktail fresh before use. The addition of benzonase rapidly hydrolyzes nucleic acids which reduces cell lysate viscosity.</li>
	</ol>
	</li>
	<li>Determine protein concentration of the samples using BCA Protein Assay Kit or use any other method which tolerates 1% SDS in solution.
	<ol>
		<li>Add 2 &#181;L of lysate and protein standards (0, 1, 2, 4, and 8 &#181;g/mL of BSA in lysis buffer) into the wells of the 96-well clear plate.</li>
		<li>Mix reagent A with reagent B at 50:1 ratio and add 200 &#181;L per well. Incubate for 20 min at 37 &#176;C and read the absorbance.</li>
	</ol>
	</li>
	<li>Adjust the protein concentration with lysis buffer to be equal across the samples.</li>
	<li>Add 20 &#181;L of 4x Loading Buffer to 60 &#181;L of cell lysate and heat at 95 &#176;C for 5 min. After heat denaturation, the lysates can be stored at -20 &#176;C.</li>
</ol>
5. Western blot analysis
<ol>
	<li>Load 5-20 &#181;g of total cell lysate for analysis of histone proteins and 20-100 &#181;g for other proteins into a 4-12% Bis-Tris protein gel.</li>
	<li>Run the gel in MOPS SDS running buffer (50 mM MOPS, 50 mM Tris Base, 0.1% SDS w/v, 1 mM EDTA, pH 7.7) for about 2 h at 100 V or until the dye front reaches the bottom of the gel.</li>
	<li>If performing a wet transfer, assemble the transfer sandwich in ice-cold Tris-Glycine transfer buffer (25 mM Tris, 192 mM Glycine, 20% v/v methanol, and 0.05% w/v SDS).
	<ol>
		<li>Place sponges, filter paper, PVDF membrane, and gel according to manufacturer&#39;s instructions. Activate PVDF membrane by soaking in methanol and equilibrate gel in transfer buffer for 30 s before assembly. 
		NOTE: Use recommended PVDF western blotting membrane since it has low autofluorescence and suitability for low molecular weight proteins, such as histones (Table of Materials).</li>
	</ol>
	</li>
	<li>Transfer proteins from the gel to PVDF membrane in Tris-Glycine transfer buffer at 70 V for 1.5 h on ice.</li>
	<li>Block membrane for 30 min in blocking buffer (5% w/v milk in phosphate-buffered saline, PBS). Rinse with wash buffer (PBST: 0.1% v/v Tween-20 in PBS), and incubate with primary antibodies in bovine serum albumin (BSA) solution&#160;(5% BSA in PBST) overnight at 4 &#176;C (Table 3). 
	NOTE: For longer storage, filter-sterilize BSA solution, add 0.02% w/v sodium azide, and keep at 4 &#176;C.</li>
	<li>Wash membrane 3 x 5 min with PBST. Then incubate with goat-anti-rabbit (IR800) and donkey anti-mouse (IR680) antibodies in recommended blocking buffer (Table of Materials, Table 3) for 30 min at RT and wash 3 x 5 min with PBST.</li>
	<li>Read the signal on a fluorescent western blot imager at 800 and 700 nm. Preferably use the instrument which allows imaging strong and faint bands clearly in a single image with high sensitivity and dynamic range, high signal-to-noise ratio, a warning when an image saturation is reached as well as multiplexing of two fluorescent colors in the same sample band.</li>
	<li>Determine band intensities for western blot analysis using appropriate software for fluorescent western imaging.</li>
</ol>

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L., 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+guide+to+modern+quantitative+fluorescent+western+blotting+with+troubleshooting+strategies.">A guide to modern quantitative fluorescent western blotting with troubleshooting strategies.</a> The Journal of Visualized Experiments. (93), e52099 (2014).</li><li>Thandapani, P., O'Connor, T. R., Bailey, T. L., Richard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+RGG/RG+motif.">Defining the RGG/RG motif.</a> Molecular Cell. 50 (5), 613-623 (2013).</li><li>Dhar, 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=Loss+of+the+major+Type+I+arginine+methyltransferase+PRMT1+causes+substrate+scavenging+by+other+PRMTs.">Loss of the major Type I arginine methyltransferase PRMT1 causes substrate scavenging by other PRMTs.</a> Science Reports. 3, 1311 (2013).</li><li>Kaniskan, H. U., 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+potent,+selective+and+cell-active+allosteric+inhibitor+of+protein+arginine+methyltransferase+3+(PRMT3).">A potent, selective and cell-active allosteric inhibitor of protein arginine methyltransferase 3 (PRMT3).</a> Angewandte Chemie International Edition. 54 (17), 5166-5170 (2015).</li><li>Eram, M. 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=A+Potent,+Selective,+and+Cell-Active+Inhibitor+of+Human+Type+I+Protein+Arginine+Methyltransferases.">A Potent, Selective, and Cell-Active Inhibitor of Human Type I Protein Arginine Methyltransferases.</a> ACS Chemical Biology. 11 (3), 772-781 (2016).</li><li>Shen, Y., 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=Discovery+of+a+Potent,+Selective,+and+Cell-Active+Dual+Inhibitor+of+Protein+Arginine+Methyltransferase+4+and+Protein+Arginine+Methyltransferase+6.">Discovery of a Potent, Selective, and Cell-Active Dual Inhibitor of Protein Arginine Methyltransferase 4 and Protein Arginine Methyltransferase 6.</a> Journal of Medicinal Chemistry. 59 (19), 9124-9139 (2016).</li><li>Nakayama, K., 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=TP-064,+a+potent+and+selective+small+molecule+inhibitor+of+PRMT4+for+multiple+myeloma.">TP-064, a potent and selective small molecule inhibitor of PRMT4 for multiple myeloma.</a> Oncotarget. 9 (26), 18480-18493 (2018).</li><li>Bonday, Z. Q., 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=LLY-283,+a+Potent+and+Selective+Inhibitor+of+Arginine+Methyltransferase+5,+PRMT5,+with+Antitumor+Activity.">LLY-283, a Potent and Selective Inhibitor of Arginine Methyltransferase 5, PRMT5, with Antitumor Activity.</a> ACS Medicinal Chemistry Letters. 9 (7), 612-617 (2018).</li><li>Szewczyk, M. 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=Pharmacological+inhibition+of+PRMT7+links+arginine+monomethylation+to+the+cellular+stress+response.">Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response.</a> Nature Communications. 11 (1), 2396 (2020).</li><li>Goulet, I., Gauvin, G., Boisvenue, S., Cote, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+yields+protein+arginine+methyltransferase+1+isoforms+with+distinct+activity,+substrate+specificity,+and+subcellular+localization.">Alternative splicing yields protein arginine methyltransferase 1 isoforms with distinct activity, substrate specificity, and subcellular localization.</a> Journal of Biological Chemistry. 282 (45), 33009-33021 (2007).</li><li>Siarheyeva, 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=An+allosteric+inhibitor+of+protein+arginine+methyltransferase+3.">An allosteric inhibitor of protein arginine methyltransferase 3.</a> Structure. 20 (8), 1425-1435 (2012).</li><li>Stefansson, O. A., Esteller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CARM1+and+BAF155:+an+example+of+how+chromatin+remodeling+factors+can+be+relocalized+and+contribute+to+cancer.">CARM1 and BAF155: an example of how chromatin remodeling factors can be relocalized and contribute to cancer.</a> Breast Cancer Research. 16 (3), 307 (2014).</li><li>Pesiridis, G. S., Diamond, E., Van Duyne, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+pICLn+in+methylation+of+Sm+proteins+by+PRMT5.">Role of pICLn in methylation of Sm proteins by PRMT5.</a> Journal of Biological Chemistry. 284 (32), 21347-21359 (2009).</li><li>Guccione, 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=Methylation+of+histone+H3R2+by+PRMT6+and+H3K4+by+an+mLL+complex+are+mutually+exclusive.">Methylation of histone H3R2 by PRMT6 and H3K4 by an mLL complex are mutually exclusive.</a> Nature. 449 (7164), 933-937 (2007).</li><li>Yudao Shen, ., L, F., 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+First-in-class,+Highly+Selective+and+Cell-active+Allosteric+Inhibitor+of+Protein+Arginine+Methyltransferase+6+(PRMT6).">A First-in-class, Highly Selective and Cell-active Allosteric Inhibitor of Protein Arginine Methyltransferase 6 (PRMT6).</a> BioRxiv. , 1-21 (2020).</li><li>Pahlich, S., Zakaryan, R. P., Gehring, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+proteins+interacting+with+protein+arginine+methyltransferase+8:+the+Ewing+sarcoma+(EWS)+protein+binds+independent+of+its+methylation+state.">Identification of proteins interacting with protein arginine methyltransferase 8: the Ewing sarcoma (EWS) protein binds independent of its methylation state.</a> Proteins. 72 (4), 1125-1137 (2008).</li><li>Lee, J., Sayegh, J., Daniel, J., Clarke, S., Bedford, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRMT8,+a+new+membrane-bound+tissue-specific+member+of+the+protein+arginine+methyltransferase+family.">PRMT8, a new membrane-bound tissue-specific member of the protein arginine methyltransferase family.</a> Journal of Biological Chemistry. 280 (38), 32890-32896 (2005).</li><li>Kim, J. D., Kako, K., Kakiuchi, M., Park, G. G., Fukamizu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EWS+is+a+substrate+of+type+I+protein+arginine+methyltransferase,+PRMT8.">EWS is a substrate of type I protein arginine methyltransferase, PRMT8.</a> International Journal of Molecular Medicine. 22 (3), 309-315 (2008).</li><li>Yang, Y., 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=PRMT9+is+a+type+II+methyltransferase+that+methylates+the+splicing+factor+SAP145.">PRMT9 is a type II methyltransferase that methylates the splicing factor SAP145.</a> Nature Communications. 6, 6428 (2015).</li><li>Rakow, S., Pullamsetti, S. S., Bauer, U. M., Bouchard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+epigenome+functions+of+PRMTs+and+their+substrates.">Assaying epigenome functions of PRMTs and their substrates.</a> Methods. 1175, 53-65 (2020).</li><li>Musiani, D., 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=Proteomics+profiling+of+arginine+methylation+defines+PRMT5+substrate+specificity.">Proteomics profiling of arginine methylation defines PRMT5 substrate specificity.</a> Science Signaling. 12 (575), (2019).</li><li>Musiani, D., Massignani, E., Cuomo, A., Yadav, A., Bonaldi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+Computational+Approaches+for+the+Large-Scale+Analysis+of+Protein+Arginine+Methylation+by+Mass+Spectrometry.">Biochemical and Computational Approaches for the Large-Scale Analysis of Protein Arginine Methylation by Mass Spectrometry.</a> Current Protein and Peptide Science. 21 (7), 725-739 (2020).</li><li>Shishkova, 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=Global+mapping+of+CARM1+substrates+defines+enzyme+specificity+and+substrate+recognition.">Global mapping of CARM1 substrates defines enzyme specificity and substrate recognition.</a> Nature Communications. 8, 15571 (2017).</li><li>Pawlak, M. 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The authors do not have any competing financial interests or other conflicting interests to declare.

Here, the detailed cellular assay protocols for members of the PRMT family are described that use fluorescent western blotting methods. Unique substrates for which the changes in arginine methylation can be easily detected upon individual PRMT loss or catalytic inhibition and cannot be compensated by other family members were selected. Some proteins are methylated by multiple PRMTs21,23, suggesting an overlap in substrate specificity where some PRMTs contribute only a small amount of cellular mark in a given protein substrate24,25,26,27, for example, both PRMT8 and PRMT1 contribute to methylation of EWS. Therefore, each assay required thorough validation of substrates and antibodies with knockdown and/or overexpression experiments and further validation with well-characterized selective inhibitors. PRMT specific substrates were identified for which methylation mark changes could be detected within 2-3 days post-PRMT loss/inhibition to avoid compounding effects of reduced cell viability and proliferation that may indirectly affect the methyl-arginine mark levels. Although it was possible to find unique substrates for PRMT1, 4, 5, 7, and 9; for PRMT3, 6, and 8 the gain of function approach had to be employed. Several arginine methyl-specific antibodies were tested for various cellular targets, but none were able to detect significant changes within 3 days of PRMT3 and PRMT6 knockdown; therefore, biomarker assays were developed using ectopically expressed enzymes together with catalytically inactive mutants, which served as a control for the baseline substrate methylation. PRMT8 is a close PRMT1 homolog and shares similar substrate preferences. As a PRMT8 selective biomarker could not be identified, an assay in PRMT1 knockdown cells was developed, where PRMT8 was co-expressed together with EWS. PRMT1 is also a major enzyme responsible for H4R3 asymmetric methylation, therefore, to use H4R3me2a as a biomarker for PRMT3 and PRMT6 cellular assays, cells with low basal H4R3me2a levels were chosen as well as catalytically inactive mutants were used as a background control. Although endogenous assays are preferred, exogenous assays prove invaluable for testing the cellular potency of several selective PRMT inhibitors7,8,9. With growing knowledge of PRMT biology, we expect to improve the assays by finding more specific biomarker proteins for PRMT3, PRMT6, and PRMT8.
The use of validated antibodies and appropriate controls are critical for the PRMT assay performance. All antibodies recommended here have been thoroughly validated by knockdown and overexpression experiments, however, batch-to-batch differences, especially in the case of polyclonal antibodies, may still influence their performance. Therefore, it is crucial to use genetic methods and chemical probes together with their closely related negative controls to confirm assay reliability. Additionally, for PRMT assays that require protein overexpression, it is crucial to use catalytically inactive mutants along with wild-type protein to determine the basal methylation levels.
This collection of quantitative assays for profiling the activity of PRMTs in cells can be broadly useful for the scientific community since it can be rapidly and easily implemented with minimal equipment and limited technical expertise, involving only basic cell culturing and fluorescent western blotting techniques. The recommended antibodies and chemical probes for PRMTs can also be utilized for activity-based protein profiling (ABPP) assays to establish the suitability of a given ABPP probe, monitor target engagement, and assess off-target effects by using the competitive ABPP format. The assay development approaches discussed here can also be extrapolated for other enzyme families such as protein lysine-methyltransferases and acetyltransferases.

Arginine methylation is an important post-translational modification that regulates protein-protein and protein-RNA interactions, thus playing an important role in various cellular processes such as pre-mRNA splicing, DNA damage, transcription response, and growth factor-mediated transduction1,2. Arginine is methylated by protein arginine methyltransferases (PRMTs) resulting in monomethyl arginine (Rme1), asymmetrical dimethylarginine (Rme2a), or symmetrical dimethylarginine (Rme2s)3. Based on the methylation type, PRMTs are classified into three groups: Type I (PRMT1, 2, 3, 4, 6, and 8), which catalyze mono- and asymmetric dimethylation; Type II (PRMT5 and PRMT9), which catalyze mono- and symmetric dimethylation; and Type III (PRMT7), which can only monomethylate arginine3.
Due to a growing number of commercially available arginine methylation-specific antibodies, PRMT activity can be measured using western blotting. Fluorescent-based western blot is the preferred technique over chemiluminescent detection due to a greater dynamic range and linearity, higher sensitivity, and allowing for multiplexing4. To quantify the protein methylation levels, normalization of the methylation signal to total protein levels is required. By choosing the antibodies for total and methylated protein raised in different host species (e.g., mouse and rabbit), secondary antibodies labeled with different fluorophores can be used and the signal for both antibodies can be determined in the same sample band. Methyl-arginine antibodies were developed to identify and characterize monomethylated, asymmetrically, or symmetrically dimethylated proteins where methyl-arginine is found in a specific context. Since the majority of PRMTs methylate glycine- and arginine-rich motifs within their substrates5, several antibodies were raised for the peptides containing monomethyl or asymmetric, symmetric dimethyl-arginine-glycine repeats such as D5A12, ASYM24, or ASYM25, and SYM11, respectively. Other methyl-arginine antibodies were generated against a peptide library containing asymmetric, symmetric dimethyl- and monomethyl arginine in a repeat context facilitating the detection of methyl-arginine in these particular contexts6. There is also an increasing number of antibodies that recognize specific arginine mark on a single protein which enable selective detection of methylation such as histone H4R3me2a or BAF155-R1064me2a.
There are several commercially available PRMT inhibitors, which can be used as tools for PRMT cellular assays. However, not all of them are thoroughly characterized for selectivity and off-target effects and some should be used with caution. The Structural Genomic Consortium, in collaboration with academic labs and pharma partners, has developed well-characterized potent, selective, and cell-permeable PRMT inhibitors (chemical probes) that can be used with no restrictions by the scientific community. Information on these inhibitors can be found on https://www.thesgc.org/chemical-probes/epigenetics and https://www.chemicalprobes.org/.&#160;Chemical probes are small-molecule inhibitors with&#160;in vitro IC50 or Kd &#60; 100 nM, over 30-fold selectivity over proteins in the same family, and significant cellular activity at 1 &#181;M. Additionally, each chemical probe has a close chemical analog that is inactive against the intended target7,8,9,10,11,12.
The goal of this protocol is to measure the cellular activity of individual PRMT family members using the fluorescent western blot method. Here detailed information on validated assay biomarkers, antibodies, and potent cell-active inhibitors as well as valuable strategies for successful assay implementation are provided.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm TC dishes</td>
			<td>Greiner bio-one</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well TC plates</td>
			<td>Greiner bio-one</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8211;12% Bis-Tris Protein Gels</td>
			<td>ThermoFisher Scientiffic</td>
			<td>NP0323BOX, NP0322BOX,NP0321BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Hybond P PVDF membrane</td>
			<td>Millipore-Sigma</td>
			<td>10600021</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Asym 24</td>
			<td>Millipore-Sigma</td>
			<td>07-414</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Asym 25</td>
			<td>Millipore-Sigma</td>
			<td>09-814</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-B-actin</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-47778</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-BAF155</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-32763</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-BAF155-R1064me2a</td>
			<td>Millipore-Sigma</td>
			<td>ABE1339</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-FLAG (#, 1:5000)</td>
			<td>Millipore-Sigma</td>
			<td>F4799</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GFP</td>
			<td>Clontech</td>
			<td>632381</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-H3</td>
			<td>Abcam</td>
			<td>ab10799</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-H3R2me2a</td>
			<td>Millipore-Sigma</td>
			<td>04-848</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-H3R8me2a</td>
			<td>Rockland</td>
			<td>600-401-I67</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-H4</td>
			<td>Abcam</td>
			<td>ab174628</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-H4R3me2a</td>
			<td>Active Motif</td>
			<td>39705</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Hsp/Hsc70</td>
			<td>Enzo</td>
			<td>ADI-SPA-820</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-PRMT1</td>
			<td>Millipore-Sigma</td>
			<td>07-404</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-PRMT3</td>
			<td>Abcam</td>
			<td>ab191562</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-PRMT4</td>
			<td>Bethyl</td>
			<td>#A300-421A</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-PRMT5</td>
			<td>Abcam</td>
			<td>ab109451</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-PRMT6</td>
			<td>Abcam</td>
			<td>ab47244</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-PRMT7</td>
			<td>Abcam</td>
			<td>ab179822</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Rme1</td>
			<td>CST</td>
			<td>8015</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Rme2a</td>
			<td>CST</td>
			<td>13522</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Rme2s</td>
			<td>CST</td>
			<td>13222</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Rme2s (ASYM25), Millipore, , 1:2000)</td>
			<td></td>
			<td>09-814</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-SAP145 (Abcam, #, 1:1000)</td>
			<td>Abcam</td>
			<td>ab56800</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-SAP145-R508me2s</td>
			<td></td>
			<td></td>
			<td>kind gift from Dr. Yanzhong Yang, Beckman Research Institute of City of Hope</td>
		</tr>
		<tr>
			<td>anti-SmBB&#8217;</td>
			<td>Santa Cruz Biotechnologies</td>
			<td>sc-130670</td>
			<td></td>
		</tr>
		<tr>
			<td>benzonase</td>
			<td></td>
			<td></td>
			<td>PRODUCED IN-HOUSE</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Millipore-Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>C2C12</td>
			<td></td>
			<td></td>
			<td>gift from Dr. Stephane Richard, McGill University</td>
		</tr>
		<tr>
			<td>cOmplete, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Millipore-Sigma</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Wisent</td>
			<td>319-005-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Bioshop</td>
			<td>DMS666.100</td>
			<td></td>
		</tr>
		<tr>
			<td>donkey anti-mouse IgG-IR680</td>
			<td>Licor</td>
			<td>926-68072</td>
			<td></td>
		</tr>
		<tr>
			<td>doxycycline</td>
			<td>Millipore-Sigma</td>
			<td>D9891</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Bioshop</td>
			<td>EDT111.500</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Wisent</td>
			<td>80150</td>
			<td></td>
		</tr>
		<tr>
			<td>glycine</td>
			<td>Bioshop</td>
			<td>GLN002.5</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-anti-rabbit IgG-IR800</td>
			<td>Licor</td>
			<td>926-32211</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T</td>
			<td></td>
			<td></td>
			<td>gift from Dr. Sam Benchimol, York University</td>
		</tr>
		<tr>
			<td>Image Studio Software ver 5.2</td>
			<td>Licor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Loading buffer: NuPAGE LDS Sample Buffer (4x)</td>
			<td>ThermoFisher Scientiffic</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr>
			<td>MCF7</td>
			<td></td>
			<td>ATCC&#174; HTB-22&#8482;</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bioshop</td>
			<td>SOD001.1</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE MOPS SDS Running Buffer</td>
			<td>ThermoFisher Scientiffic</td>
			<td>NP0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Blocking Buffer (dilute 4 x with PBST)</td>
			<td>Licor</td>
			<td>927-40000</td>
			<td>Intercept (PBS) Blocking Buffer can also be used # 927-70001</td>
		</tr>
		<tr>
			<td>Odyssey CLX Imaging System</td>
			<td>Licor</td>
			<td>model number 9140</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (tissue culture)</td>
			<td>Wisent</td>
			<td>311-010-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (western blot)</td>
			<td>Bioshop</td>
			<td>PBS405.4</td>
			<td></td>
		</tr>
		<tr>
			<td>penstrep</td>
			<td>Wisent</td>
			<td>450-201-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce&#8482; BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientiffic</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Bioshop</td>
			<td>SDS001.1</td>
			<td></td>
		</tr>
		<tr>
			<td>skim milk powder</td>
			<td>Bioshop</td>
			<td>SKI400.500</td>
			<td></td>
		</tr>
		<tr>
			<td>TC20 automated cell counter</td>
			<td>Biorad</td>
			<td>1450102</td>
			<td></td>
		</tr>
		<tr>
			<td>Tripsin-EDTA (0.25%)</td>
			<td>Wisent</td>
			<td>325-043-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Bioshop</td>
			<td>TRS003.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Tritton X-100</td>
			<td>Bioshop</td>
			<td>TRX506</td>
			<td></td>
		</tr>
		<tr>
			<td>trypan blue</td>
			<td>GIBCO</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Bioshop</td>
			<td>TWN510.500</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative methods to study protein arginine methyltransferase 1-9 activity in cells

Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine members that can monomethylate or symmetrically/asymmetrically dimethylate arginine residues. Several antibodies recognizing different types of arginine methylation of various proteins are available; thus, providing tools for the development of PRMT activity biomarker assays. PRMT antibody-based assays are challenging due to overlapping substrates and motif-based antibody specificities. These issues and the experimental setup to investigate the arginine methylation contributed by individual PRMTs are discussed. Through the careful selection of the representative substrates that are biomarkers for eight out of nine PRMTs, a panel of PRMT activity assays were designed. Here, the protocols for cellular assays quantitatively measuring the enzymatic activity of individual members of the PRMT family in cells are reported. The advantage of the described methods is their straightforward performance in any lab with cell culture and fluorescent western blot capabilities. The substrate specificity and chosen antibody reliability were fully validated with knockdown and overexpression approaches. In addition to detailed guidelines of the assay biomarkers and antibodies, information on the use of an inhibitor tool compound collection for PRMTs is also provided.

Watch this Scientific Journal Video about Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells at JoVE.com

Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells

These protocols provide the methodology used to assess the enzymatic activity of individual members of the protein arginine methyltransferase (PRMT) family in cells. Detailed guidelines on assessing PRMT activity using endogenous and exogenous biomarkers, methyl-arginine recognizing antibodies, and inhibitor tool compounds are described.

Protein methyltransferases (PRMTs) catalyze the transfer of a methyl group to arginine residues of substrate proteins. The PRMT family consists of nine ...

quantitative-methods-to-study-protein-arginine-methyltransferase-1-9

Research

JoVE Journal

Biology

4.0K Views.  University of Toronto. These protocols provide the methodology used to assess the enzymatic activity of individual members of the protein arginine methyltransferase (PRMT) family in cells. Detailed guidelines on assessing PRMT activity using endogenous and exogenous biomarkers, methyl-arginine recognizing antibodies, and inhibitor tool compounds are described.

Video: Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells

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.

Badania wizualne Wrażliwość na prędkość i kierunek ruchu w Jaszczurki

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

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...