Name: quantitative methods to study protein arginine methyltransferase 1-9 activity in cells
Uploaded: 2021-08-07T13:30:00
Duration: 8 min 11 s
Description: Watch this Scientific Journal Video about Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells at JoVE.com

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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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. 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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 ...

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