Typicalmodificationsites:RGGboxorRXRsequencemotifsR-arginine,G-glycine,X-anyaminoacid.Enzymescatalysingproteinargininemethylation:PRMT1fromratandthehumanhomologueHRMT1L2,humanPRMT2(HRMT1L1),ratandhumanPRMT3,mouseCARM1(PRMT4),humanJBP1(PRMT5).SubstratesofPRMTs:InmammaliancellshnRNPscontainabout65%ofthetotalNG,NG-dimethylargininefoundinthecellnucleus.Additionally,functionallydiverseproteinsaresubstratesforPRMTfamilymembers,includingfibrillarinandnucleolin,involvedinribosomalbiogenesis,histones,fibroblastgrowthfactor,interleukinenhancer-bindingfactor3theSRCkinaseadapterproteinSam68,STAT1,andEWS.Analysisofproteinargininemethylationinvivo.1.Propagatecellsofinterestinnormalmediumcorrespondingtothecelltype(fortypicaladherentcellsDMEM,10%FCS,antibiotics).Foradherentcellscelldensityontheexperimentdayshouldbe50-70%.2.Washcells2xwithPBSandreplacethemediumwithmediumdeficientinmethionine(ormethionineandcysteine).Forstandardadherentcells-MEMEaglemediumwithoutmethionine(GibcoBRL),supplementedwithpenicillin,streptomycinand5%dialysedfoetalcalfserumfor30min.3.Inhibittranslationbyaddingtothemediumacycloheximide/chloramphenicolmixtureinafinalconcentrationof100μg/mland40μg/ml(no-translationmix,NTM),respectively.4.Incubatedcellswithtranslationinhibitorsfor1hbeforelabellingaswellasduringthelabellingprocedure.Todetectmethylatedproteinsaddthemethyl-groupdonorL-[mehtyl-3H]methionine(Amersham)inafinalofconcentration30μCi/ml.5.Monitortheproteintranslationinhibitionbycelllabellingwith35S-methionine(ICN)20μCi/mlinparallelcontrolplates+/-NTM.6.Continuemetaboliccelllabellingfor3.5h.7.Washcells2xwithPBS,harvestandlyseinbufferyoulike.Forexamplemiddlestringencybuffersufficientforlysesofadherentcells:NP40-1%lysisbuffer(50mMTRIS-HClpH8.0,150mMNaCl,1%NP-40)supplementedwithaproteaseinhibitorcocktailtablet(Roche).8.Identifytheproteinofinterestbyimmunoprecipitation(IP)withspecificantibody.SuggestedIPprotocol.a)PreclearcelllysateswithproteinG-sepharose.b)Incubate200μgofpre-clearedlysatesovernightwithproteinGcoupledtothecorrespondingantibody(rotationovernight,4°C,totalIPvolume1.5ml)c)Washcomplexes5timeswithcorresponding(forexampleNP40-1%buffer).Keepsamplesat4°Cduringwashingstepsd)AddofSDS-loADIngbufferwereaddedtoeachsample(optimal40-60μl).Heatsamplesat95°Cfor5minandanalysebySDS-PAGE.Incubatedfor1hwithH3enhancer,forexampleEnlightsolution(EnerGene),dryandexposetoKodakXOMAT(AR)filmat–80°C.Donotforgetimportantcontrols!CelllysedshouldbeanalysedinPAGEinparallel,andnosignalshouldbeobservedinsamplestreatedbyNTMandlabelledby35S-methionineFormoredetailsaboutprocedureandcontrolssee:KzhyshkowskaJetal,HeterogeneousnuclearribonucleoproteinE1B-AP5ismethylatedinitsArg-Gly-Gly(RGG)boxandinteractswithhumanargininemethyltransferaseHRMT1L1.BiochemJ.2001Sep1;358(Pt2):305-14.

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