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1、RESEARCHARTICLESaS,wereobtainedfromP.T.Lansbury[J.C.RochetetwerepuriÞedonnickelNTA-agarose(QIAGEN).En-36.J.Q.Trojanowski,M.Goedert,T.Iwatsubo,V.M.Lee,al.,Biochemistry39,10619(2000)].zymaticactivitywastestedinUbshiftassays.CellDeathDiffer.5,823(1999).21
2、.Thesilver-stainedbandat22kDinanHP2Aimmu-29.Parkinwasimmunoprecipitated(yieldingIP-parkin)37.M.S.Goldberg,P.T.Lansbury,NatureCellBiol.2,noprecipitateofnormalhumanbrainandaco-mi-fromfrontalcortexhomogenatesorHEK293cellsE115(2000).gratingsilver-negatives
3、licefromanadjacentlaneoftransientlytransfectedwithmyc-parkincDNA(1038.N.F.Bence,R.M.Sampat,R.R.Kopito,Science292,aparkin-preabsorbedHP2Aprecipitatewereexcised,mg)(13).IPparkinwasincubatedat37¡Cin50mlof1552(2001).trypsindigestedandsubjectedtoblindanalys
4、isbyreactionbuffercontainingATP(4mMATPin50mM39.C.J.Cummingsetal.,Neuron24,879(1999).massspectrometryatMDSProteomics,Inc.(Toronto,Tris-HCl,pH7.5,2mMMgCl2),100ngofrecombi-40.L.Wells,K.Vosseller,G.W.Hart,Science291,2376ON)[A.Shevchenkoetal.,Anal.Chem.68,8
5、50nanthumanE1,2mgofUbcH7(E2),and2mgHis-Ub(2001).(1996)].TheHP2A-speciÞc22-kDproteinyielded(allfromAfÞnityResearchProducts,Exeter,UK).The41.WethankH.Mori,Y.Mizutani,andK.YamaneforARPDtrypticpeptidescorrespondingtoaa13Ð21,44Ð58,reactionwasterminatedbyadd
6、ing20mlof4Xbrainspecimens;J.Chanforprovidingnormalhuman46Ð58,59Ð80,61Ð80,81Ð96,and81Ð97ofhumansamplebuffer,and25mlaliquotsofthereactionbrain;andthepatientsÕfamiliesforthedonationofaS(GenBankaccession#L08850),eachendingwithmixtureswereelectrophoresedand
7、immunoblotted.tissue.WealsothankK.WynnandP.Howleyforalysine,asexpected.NoaSsequencewasdetected30.WepreparedWTandARPD-mutantparkincDNAs[N.anti-E6-AP(JH-16),T.SuzukiandK.Tanakaforaparkinintheco-migratingslicefromthepreabsorbedlane.Hattorietal.,Biochem.Bi
8、ophys.Res.Commun.249,cDNA-containingvector,P.Lansburyforreagentsand22.Y.Mizuno,N.Hattori,H.Mori,Biomed.Pharmaco-754(1998);L.Terrenietal.,Neurology56,463criticalreviewofourmanuscript,andM.Medinaforther.53,109(1999).(2001)]inapcDNA3.1(1)v
