Molecular Characterisation of Endogenous Vangl2Vangl1 Heteromeric Protein Complexes 英文参考文献.docVIP

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Molecular Characterisation of Endogenous Vangl2Vangl1 Heteromeric Protein Complexes 英文参考文献.doc

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Molecular Characterisation of Endogenous Vangl2Vangl1 Heteromeric Protein Complexes 英文参考文献

MolecularCharacterisationofEndogenousVangl2/ Vangl1HeteromericProteinComplexes EdwigeBelotti1,2,3,4.,TaniaM.Puvirajesinghe1,2,3,4.,Ste′phaneAudebert1,2,3,4,EmilieBaudelet1,2,3,4 LucCamoin1,2,3,4,MichelPierres5,LeaLasvaux6,Ge′raldineFerracci7,MireilleMontcouquiol6 ,Jean- , PaulBorg1,2,3,4 * 1Inserm U1068, CRCM, Marseille, France, 2CNRS UMR7258, CRCM, Marseille, France, 3Institut Paoli-Calmettes, Marseille, France, 4Aix-Marseille Universite′, Marseille, France,5Centred9ImmunologiedeMarseille-Luminy,UMR6102INSERM/CNRS,Marseille,France,6NeurocentreMagendie,PlanarPolarityandPlasticityGroup,INSERM U862,BordeauxUniversite′,Bordeaux,France,7Aix-MarseilleUniversite′, CNRS,CRN2MUMR7286,Marseille,France Abstract Background:MutationsinthePlanarCellPolarity(PCP)coregeneVangl2causethemostsevereneuraltubedefects(NTD) inmiceandhumans.GeneticstudiesshowthattheVangl2genegeneticallyinteractswithaclosehomologueVangl1 .How preciselyVangl2andVangl1proteinsinteractandcrosstalkhasremainedadifficultissuetoaddress,withthemainobstacle being the accurate discrimination of the two proteins, which share close sequence homology. Experimental evidence previously presented has been sparse and addressed with ectopically expressed proteins or with antibodies unable to biochemicallydiscriminateVangl1fromVangl2,thereforegivingrisetounclearresults. MethodologyandMainFindings:Ahighlyspecificmonoclonalanti-Vangl2antibodywasgeneratedandrigorouslytested on both recombinant and extracted Vangl2 using surface plasmon resonance (SPR) analysis, western blot, and immunoprecipitation experiments. This antibody efficiently affinity-purified Vangl2 from cell lysates and allowed the unambiguous identification of endogenous Vangl2 by proteomic analysis. Vangl1 was also present in Vangl2 immunoprecipitates, establishing the first biochemical evidence for the existence of Vangl2/Vangl1 heterodimers at an endogenouslevel.Epitope-taggedVangl2andVangl1confirmedthatbothproteinsinteractandcolocalizeattheplasma memb