PreparationofNuclearExtractsforGelShiftsandWesternsBlots Thefollowingprotocolisoptimizedforabout107cells(nearconfluent10cmplate).NOTE:ALLCOMPONENTS(BUFFERS,PROTEASEINHIBITORS,ETC)MUSTBEKEPTONICEDURINGTHEENTIREPROCEDURE. 1.WashplateswithPBStwotimes. 2.Prepare5mlofBufferAina15mlconicalc’fugetube,andaddthefollowing: -5ulofeachproteaseinhibitor(leupeptin,PMSF,aprotinin,pepstatin,andDTT) -200ulof10%IGEPAL. Add0.5mlofthispreparationdirectlytoeachplate,andwait10min.atroomtemp. 3.ScrapecellswithNEWsterilescraper,thenpipetupanddownwithP1000severaltimestodisruptcellclumps.NOTE:youwillhavenearly1mloflysateatthispoint. 4.Transferthislysatetoa1.5microcentrifugetube,placeonice. 5.Centrifugeat4Cattopspeed(15000xg)for3min. 6.Placetubesonice. 7.Savesupernatant(cytosolicfraction)forluciferase/ß-galassay,ifdesired.Otherwise,discardthesupernatant. 8.Prepare1mlofBufferBina1.5mlEppendorf,andadd1ulofeachproteaseinhibitorasabove,butthistimeDONOTaddIGEPAL.Add150mlofthisbuffertoeachtube,andresUSPendpelletbypipetingupanddownwithaP200.Placeonice. 9.Shakevigorouslyat4Cfor2h. 10.Centrifugeat4C,topspeedfor5min.MeasureBradford(protein)concentrationusing5ulofeachsample,thenaliquot15ulaliquotsintoprechilled0.5mlprelabeledmicrocentrifugetubes,freezeinliquidnitrogen,andstorein–80Cfreezer. BufferA: 10mMHEPES,pH7.9 10mMKCl 0.1mMEDTA Addjustbeforeuse:1mMDTT,0.5mMPMSF,5ulof10ug/ulofaprotinin,leupeptin,andpepstatinAto5mlofbuffer. BufferB: 20mMHEPES,pH7.9 0.4MNaCl 1mMEDTA 10%Glycerol Addjustbeforeuse:DTT,PMSF,aprotinin,leupeptin,pepstatinAasabove.