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Bpsbioscience/CSF1R / SRE Reporter Kit (MAPK/ERK Signaling Pathway)/79379/500 reactions
ColonyStimulatingFactor1Receptor(CSF1R,CD115,M-CSF-R)isthereceptorforthecytokineCSF1(MCSF;CSF-1),whichcontrolsthegrowth,function,anddifferentiationofmacrophages.ThisinteractiondrivesthedevelopmentandsurvivalofTumor-AssociatedMacrophages(TAMs),whichinturnsuppressthelocalimmuneresponsetothecancer.AnalternativeligandisIL-34,whichhasbeenimplicatedinneuroprotectiveandsurvivalsignalsfollowingbraininjuryandneurodegeneration.
Asatyrosinekinasetransmembranereceptor,activationofCSF1Rbyitsligandinitiatesavastarrayofintracellularactivity,includingactivationoftheMAPK/ERKsignalingpathway.WhenphosphorylatedbyERK,Elk1formsacomplexwithSerumResponseFactor(SRF)andbindstoSerumResponseElement(SRE),resultingintheexpressionofnumerousmitogen-inducIBLegenes.
TheCSF1RSREReporterKitisdesignedformonitoringtheactivityoftheCSF1Rsignalingpathwayinculturedcells.Thekitcontainsatransfection-readyvectorforCSF1RandSREluciferasereportervector.Uponligandbinding,activeCSF1RwillinitiatetheMAPK/ERKsignalingpathway,leADIngtoexpressionoftheSRE-controlledreporter.ThisreportercontainsthefireflyluciferasegeneunderthecontrolofmultimerizedSREresponsiveelementslocatedupstreamofaminimalpromoter.TheSREreporterispremixedwithaconstitutively-expressingRenillaluciferasevectorthatservesasaninternalcontrolfortransfectionefficiency.
Thekitalsoincludesanon-induciblefireflyluciferasevectorpremixedwithconstitutively-expressingRenillaluciferasevectorasanegativecontrol.Thenon-inducibleluciferasevectorcontainsthefireflyluciferasegeneunderthecontrolofaminimalpromoter,withoutanyadditionalresponseelements.Thenegativecontroliscriticalfordeterminingpathway-specificeffectsandthebackgroundluciferaseactivity.
•Mammaliancelllineandappropriatecellculturemedium
•96-welltissuecultureplateor96-welltissueculture-treatedwhiteclear-bottomassayplate
•Transfectionreagentformammaliancellline[WeuseLipofectamine™2000(Invitrogen#11668027)+Opti-MEMIReducedSerumMedium(Invitrogen#31985-062).However,othertransfectionreagentsworkequallywell.]
•RecombinantHumanCSF1(BPSBioscience,#90215-B)orRecombinantHumanIL34(Biolegend,#577904)
•Lowserum(0.5%FBS)growthmediumoptimizedforparentalcelltype
ForHEK293celllinesuse
BPSMedium8(BPSBioscience,#79385):MEMmedium(Hyclone#SH30024.01)supplementedwith0.5%FBS(Invitrogen#26140-079),1%non-essentialaminoacids(Hyclone#SH30238.01),1mMNapyruvate(Hyclone#SH30239.01),1%Penicillin/Streptomycin(HycloneSV30010.01)
•DualLuciferaseAssaySystem:
DualLuciferase(Firefly-Renilla)AssaySystem(BPSBioscience,#60683):Thissystemassayscellsdirectlyingrowthmedium.Itcanbeusedwithanyluminometer.Automatedinjectorsarenotrequired.
•Luminometer
•MonitorCSF1R/MAPK/ERKsignalingpathwayactivityandSRF-mediatedactivity.
•ScreenforactivatorsorinhibitorsoftheCSF1R/MAPK/ERKsignalingpathway.
•StudyeffectsofRNAiorgeneoverexpression.
1.Cannarile,MichaelA.,etal.“Colony-StimulatingFactor1Receptor(CSF1R)InhibitorsinCancerTherapy.”J.Immunother.Cancer,5(1):53(2017)
2.Liu,Yang,andXuetaoCao.“TheOriginandFunctionofTumor-AssociatedMacrophages.”CellularMolec.Immunology,12(1):1–4(2014).
3.Luo,Jian,etal.“Colony-StimulatingFactor1Receptor(CSF1R)SignalinginInjuredNeuronsFacilitatesProtectionandSurvival.”J.Exp.Med.,210(1):157–172(2013).
4.Yao,G.-Q.,etal.“CSF-1InducesFosGeneTranscriptionandActivatestheTranscriptionFactorElk-1inMatureOsteoclasts.”Calc.TissueInt.,76(5):371–378(2005).
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