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1、Chromatinimmunoprecipitation(ChIP)inhumanBcellsToevaluateMycbindingtohumanAURKAandAURKBthehumanBcellline,P493-6,wasculturedwithtetracycline(0.1ug/ml)for72hours(+Tet)andreleasedfromtetracyclinefor8hours(-Tet).Chromatin7from1.3×10cells/conditionwascross-linked,isolated,sonicateda
2、ndreversecrosslinked(Lee,Johnstoneetal.2006).Mycproteinwasimmunoprecipitatedfromsonicatedchromatinusingarabbitpolyclonalantibody(SantaCruz)andmagneticproteinGbeads(ActiveMotif,Carlsbad,CA).1:100volumeofchromatinwasprocessedwithoutantibodyandusedastotalinputcontrolforqRT-PCR.qRT
3、-PCRwasrunonimmunoprecipitatedandcontrolchromatinusingprimersdesignedtodetectE-boxesinthehumanAURKAandAURKBgenes.PrimerstodetecttheknownMyctargetgeneCCND2wererunaspositivecontrolsforthehumanMycChIP(Fernandez,Franketal.2003).PercenttotalchromatinCtinput–CtChIPimmunoprecipitatedw
4、ascalculatedusingtheequation%total=2X%inputusedforChIP.RNApreparationandanalysesExpressionprofilingwasperformedusingdatapreviouslyaccumulated(Keller,Nilssonetal.2005;Nilsson,Kelleretal.2005).Briefly,RNAwaspreparedusingtheRNeasykit(Qiagen,Valencia,CA).cRNAwassynthesizedusingtheO
5、ne-CycleTargetLabelingandControlReagentpackage(AffymetrixInc.,SantaClara,CA)andthereactionwasprobedtothe430AmouseAffymetrixchip(AffymetrixInc.).ThescanneddataoutputwasimportedintotheSpotfiresoftware(TIBCOSpotfire,Somerville,MA).Followingnormalization,selectedprobesetsofthegenes
6、indicatedinSupplementalFigureS1wereclusteredusingtheHierarchicalClusteringfunctionofSpotfire(TIBCOSpotfire).Fluorescenceinsituhybridization(FISH)FISHwasperformedusingdual-colourAURKAandAURKBprobes(SupplementalTableS1)labeledwithPlatinumBright550andPlatinumBright495(KreatechDiag
7、nostics,Amsterdam,Netherlands).8-µmparaffinsectionsweredeparaffinizedandpretreatedwith0.1%proteinaseK(Invitrogen)for15–18minat37°C.Sampleswereagedin0.1%NP-40/2×SSCandDNAwasdenaturedbytreatmentin70%formamide/2×SSC.Afterdehydrationslideswerehybridizedwith8μloftheprobesovernightat
8、37°Candwashed.Nucleiwerecounterstainedwith4,6-diamino-
