Dimerization of ABCG2 Analysed by Bimolecular Fluorescence Complementation 英文参考文献.docVIP

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Dimerization of ABCG2 Analysed by Bimolecular Fluorescence Complementation 英文参考文献.doc

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Dimerization of ABCG2 Analysed by Bimolecular Fluorescence Complementation 英文参考文献

DimerizationofABCG2AnalysedbyBimolecular FluorescenceComplementation AmeenaJ.Haider,DeborahBriggs,TimJ.Self,HannahL.Chilvers,NicholasD.Holliday,IanD.Kerr* SchoolofBiomedicalSciences,UniversityofNottingham,Nottingham,UnitedKingdom Abstract ABCG2isoneofthreehumanATPbindingcassettetransportersthatarefunctionallycapableofexportingadiverserangeof substratesfromcells.ThephysiologicalconsequenceofABCG2multidrugtransportactivityinleukaemia,andsomesolid tumours is the acquisition of cancer multidrug resistance. ABCG2 has a primary structure that infers that a minimal functional transporting unit would be a homodimer. Here we investigated the ability of a bimolecular fluorescence complementationapproachtoexamineABCG2dimers,andtoprobetheroleofindividualaminoacidsubstitutionsindimer formation. ABCG2 was tagged with fragments of venus fluorescent protein (vYFP), and this tagging did not perturb traffickingorfunction.Co-expressionoftwoproteinsbearingN-terminalandC-terminalfragmentsofYFPresultedintheir associationanddetectionofdimerizationbyfluorescencemicroscopyandflowcytometry.PointmutationsinABCG2which may affect dimer formation were examined for alterations in the magnitude of fluorescence complementation signal. Bimolecularfluorescencecomplementation(BiFC)demonstratedspecificABCG2dimerformation,butnochangesindimer formation,resultingfromsingleaminoacidsubstitutions,weredetectedbyBiFCanalysis. Citation:HaiderAJ,BriggsD,SelfTJ,ChilversHL,HollidayND,etal.(2011)DimerizationofABCG2AnalysedbyBimolecularFluorescenceComplementation.PLoS ONE6(10):e25818.doi:10.1371/journal.pone.0025818 Editor:HendrikW.vanVeen,UniversityofCambridge,UnitedKingdom ReceivedJuly1,2011;AcceptedSeptember11,2011;PublishedOctober3,2011 Copyright: ? 2011 Haider et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding:Fundingforthisst