ChIPresultsobtainedwiththeantibodydirectedagainstHDAC1
ChIPwasperformedwiththeantibodyagainstHDAC1(Cat.No.25287)onshearedchromatinfrom4,000,000HeLacells.Anantibodytitrationconsistingof1,2,5and10μgperChIPexperimentwasanalysed.IgG(2μg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersspecificfortheEIF4A2andGAPDHpromoters,usedaspositivecontrols,andfortheMYOD1geneandSat2satelliterepeat,usedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

ChIP-seqresultsobtainedwiththeantibodydirectedagainstHDAC1
ChIPwasperformedonshearedchromatinfrom4,000,000HeLacellsusing2μgoftheantibodyagainstHDAC1(Cat.No.25287)asdescribedabove.TheimmunoprecipitatedDNAwassubsequentlyanalysedonanIlluminaHiSeq2000.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer’sinstructions.The50bptagswerealignedtothehumangenomeusingtheBWAalgorithm.Figure2showsthepeakdistributionalongthecompletesequenceanda1MbregionoftheX-chromosome(figure2AandB)andintworegionssurroundingtheGAPDHandEIF4A2positivecontrolgenes,respectively(figure2CandD).

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionofantibodydirectedagainstHDAC1(Cat.No.25287),crudeserumandflowthrough.Theplateswerecoatedwiththepeptideusedforimmunizationoftherabbit.Byplottingtheabsorbanceagainsttheantibodydilution(Figure2),thetiteroftheantibodywasestimatedtobe1:75,000.

WesternblotanalysisusingtheantibodydirectedagainstHDAC1
Wholecellextracts(25μg,lane1)andnuclearextracts(25μg,lane2)fromHeLacellswereanalysedbyWesternblotusingtheantibodyagainstHDAC1(Cat.No.25287)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright(expectedsize:55kDa);theMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingtheantibodydirectedagainstHDAC1
HeLacellswerestainedwiththeantibodyagainstHDAC1(Cat.No.25287)andwithDAPI.Cellswerefixedwith4%formaldehydefor10minutesandblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabelledwiththeHDAC1antibody(left)diluted1:500inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.

Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.