Thestress-activatedproteinkinase/c-junN-terminalkinase(SAPK/JNK)familyofproteinsincludesmitogen-activatedproteinkinases(MAPKs)thatareactivatedbystress,inflammatorycytokines,mitogens,oncogenes,andinducersofcelldifferentiationandmorphogenesis.UponactivationoftheSAPK/JNKpathway,MAPKinaseKinasesphosphorylateandactivateJNKs.TheactivatedJNKstranslocatetothenucleuswheretheyphosphorylateandactivatetranscriptionfactorssuchasc-Jun.Theactivatedc-Junformshomodimersorheterodimerswithfosfamilyproteinswhichbindtotheactivatorprotein-1(AP1)responseelementandinducetargetgenetranscription.
TheAP1TransientPackisdesignedtoprovidethetoolsnecessaryfortransientlytransfectingandmonitoringtheactivityoftheJNKsignalingpathwayandthetranscriptionalactivityofAP1inculturedHEK293cells.Thekitcontainstransfection-readyvectorscontainingfireflyluciferaseasaJNKpathway-responsivereporterandconstitutivelyexpressingRenillaluciferaseasatransfectioncontrol. ItalsoincludestheDualLuciferasedetectionreagentstodetectbothluciferaseactivitiesandspecializedmediumforgrowingandassayingHEK293cells.
ThekeytotheAP1TransientPackistheAP1luciferasereportervector.ThisreportercontainsthefireflyluciferasegeneunderthecontrolofmultimerizedAP1responsiveelementslocatedupstreamofaminimalpromoter.TheAP1reporterispremixedwithaconstitutively-expressingRenillaluciferasevectorthatservesasaninternalcontrolfortransfectionefficiency.
Thepackalsoincludesanon-inducIBLefireflyluciferasevectorpremixedwithconstitutively-expressingRenillaluciferasevectorasanegativecontrol.Thenon-inducibleluciferasevectorcontainsthefireflyluciferasegeneunderthecontrolofaminimalpromoter,withoutanyadditionalresponseelements.Thenegativecontroliscriticalfordeterminingpathway-specificeffectsandthebackgroundluciferaseactivity.
Additionally,thepackincludescellculturemedium(BPSMedium1)thathasbeenoptimizedforusewithHEK293andHeLacells*.BPSMedium1includesMEMmedium,10%fetalbovineserum,1%non-essentialaminoacids,sodiumpyruvate,and1%Pen/Strep. Finally,thepackprovidestheDualLuciferase(Firefly-Renilla)AssaySystem.Theseluciferasereagentsprovidehighlysensitive,stabledetectionoffireflyluciferaseactivityandRenillaluciferaseactivity. Thedualluciferasereagentscanbeuseddirectlyincellsingrowthmedium,andcanbedetectedwithanyluminometer;automatedinjectorsarenotrequired.
*Note: thekitmaybeusedwithothercelllinesthanHEK293orHeLa,butanalternatecellculturemediummayberequiredforoptimalcellgrowth,
•ScreenforactivatorsorinhibitorsoftheJNKsignalingpathway.
•StudyeffectsofRNAiorgeneoverexpressionontheactivityoftheJNKpathway.
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