Typical modification sites: RGG box or RXR sequence motifs R-arginine, G-glycine, Enzymes catalysing protein arginine methylation: PRMT1 from rat and the human homologue HRMT1L2, human PRMT2 (HRMT1L1), rat and human PRMT3, mouseCARM1 (PRMT4), human JBP1 (PRMT5).
Substrates of PRMTs: In mammalian cells hnRNPs contain about 65% of the total NG,NG-dimethylarginine found in the cell nucleus. Additionally, functionally diverse proteinsare substrates for PRMT family members, including fibrillarin and nucleolin, involved inribosomal biogenesis, histones, fibroblast growth factor, interleukin enhancer-binding factor 3the SRC kinase adapter protein Sam68, STAT1, and EWS.
PRMT - Protein aRginine MethylTransferase Analysis of protein arginine methylation in vivo.
1. Propagate cells of interest in normal medium corresponding to the cell type (for typical adherent cells DMEM, 10% FCS, antibiotics). For adherent cells celldensity on the experiment day should be 50-70%.
2. Wash cells 2x with PBS and replace the medium with medium deficient in methionine (or methionine and cysteine). For standard adherent cells - MEMEagle medium without methionine (GIBCO BRL), supplemented with penicillin,streptomycin and 5% dialysed foetal calf serum for 30 min.
3. Inhibit translation by adding to the medium a cycloheximide/chloramphenicol mixture in a final concentration of 100 µg/ml and 40 µg/ml (no-translation mix,NTM), respectively.
4. Incubated cells with translation inhibitors for 1h before labelling as well as during the labelling procedure. To detect methylated proteins add the methyl-group donorL-[mehtyl-3H]methionine (Amersham) in a final of concentration 30 µCi/ml.
5. Monitor the protein translation inhibition by cell labelling with 35S-methionine (ICN) 20 µCi/ml in parallel control plates +/- NTM.
6. Continue metabolic cell labelling for 3.5 h.
7. Wash cells 2x with PBS, harvest and lyse in buffer you like. For example middle stringency buffer sufficient for lyses of adherent cells: NP40-1% lysis buffer (50mM TRIS-HCl pH 8.0, 150 mM NaCl, 1% NP-40) supplemented with a proteaseinhibitor cocktail tablet (Roche).
8. Identify the protein of interest by immunoprecipitation (IP) with specific antibody.
a) Preclear cell lysates with protein G-sepharose.
b) Incubate 200µg of pre-cleared lysates overnight with protein G coupled to the corresponding antibody (rotation overnight, 4°C, total IP volume 1.5 ml) c) Wash complexes 5 times with corresponding (for example NP40-1% buffer).
Keep samples at 4°C during washing steps d) Add of SDS-loading buffer were added to each sample (optimal 40-60 µl). Heat samples at 95°C for 5 min and analyse by SDS-PAGE. Incubated for 1 h with H3enhancer, for example Enlight solution (EnerGene), dry and expose to Kodak X-OMAT (AR) film at –80°C.
Cell lysed should be analysed in PAGE in parallel, and no signal should be observed in samples treated by NTM and labelled by 35S-methionine For more details about procedure and controls se


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