TheMAPK/ERKsignalingpathwayisamajorparticipantintheregulationofcellgrowthanddifferentiation.Itcanbeactivatedbyvariousextracellularstimuliincludingmitogens,growthfactors,andcytokines.Uponstimulation,MEK1/2phosphorylateandactivateERK1/2.TheactivatedERKtranslocatestothenucleuswhereitphosphorylatesandactivatestranscriptionfactors.TheTCFs(TernaryComplexFactors),includingElk1,areamongthebest-characterizedtranscriptionfactorsubstratesofERK.WhenphosphorylatedbyERK,Elk1formsacomplexwithSerumResponseFactor(SRF)andbindstoSerumResponseElement(SRE),resultingintheexpressionofnumerousmitogen-inducIBLegenes.
TheSREReporterKitTransientPackisdesignedtoprovidethetoolsnecessaryfortransientlytransfectingandmonitoringtheactivityoftheERKsignalingpathwayandthetranscriptionalactivityofSREinculturedHEK293cells.Thekitcontainstransfection-readyvectorscontainingfireflyluciferaseasaMAPK/ERKpathway-responsivereporterandconstitutivelyexpressingRenillaluciferaseasatransfectioncontrol. ItalsoincludestheDualLuciferasedetectionreagentstodetectbothluciferaseactivitiesandspecializedmediumforgrowingandassayingHEK293cells.
ThekeytotheSRETransientPackistheSREluciferasereportervector,whichisanERKpathway-responsivereporter.ThisreportercontainsthefireflyluciferasegeneunderthecontrolofmultimerizedSREresponsiveelementslocatedupstreamofaminimalpromoter.TheSREreporterispremixedwithaconstitutively-expressingRenillaluciferasevectorthatservesasaninternalcontrolfortransfectionefficiency.
Thepackalsoincludesanon-induciblefireflyluciferasevectorpremixedwithconstitutively-expressingRenillaluciferasevectorasanegativecontrol.Thenon-inducibleluciferasevectorcontainsthefireflyluciferasegeneunderthecontrolofaminimalpromoter,withoutanyadditionalresponseelements.Thenegativecontroliscriticalfordeterminingpathway-specificeffectsandthebackgroundluciferaseactivity.
Additionally,thepackincludescellculturemedium(BPSMedium1)thathasbeenoptimizedforusewithHEK293cells*.BPSMedium1includesMEMmedium,10%fetalbovineserum,1%non-essentialaminoacids,sodiumpyruvate,and1%Pen/Strep. Finally,thepackprovidestheDualLuciferase(Firefly-Renilla)AssaySystem.Theseluciferasereagentsprovidehighlysensitive,stabledetectionoffireflyluciferaseactivityandRenillaluciferaseactivity. Thedualluciferasereagentscanbeuseddirectlyincellsingrowthmedium,andcanbedetectedwithanyluminometer;automatedinjectorsarenotrequired.
*Othermammaliancelllinesmayalsobeused,butanalternatecellculturemediummayberequiredforoptimalcellgrowth.
•ScreenforactivatorsorinhibitorsoftheMAPK/ERKsignalingpathway.
•StudyeffectsofRNAiorgeneoverexpressionontheactivityoftheMAPK/ERKpathway.
2.Treisman,R.(1992)Theserumresponseelement.TrendsBiochemSci.17(10):423-426.
