NF-κB(NuclearFactor-KappaB)isaheterodimericproteincomposedofdifferentcombinationsofmembersoftheRelfamilyoftranscriptionfactors.TheNF-κB/Relfamilyoftranscriptionfactors(p50,p65,c-Rel,etc.)areinvolvedinstress,immune,andinflammatoryresponses.Inunstimulatedcells,theNF-κBdimersaresequesteredinthecytoplasmbyinhibitoryIκBproteins.Proinflammatorycytokines,LPS,growthfactors,andantigenreceptorsactivateIκBkinase(IKK),whichphosphorylatestheIκBproteins.PhosphorylationofIκBleadstoitsdegradation,freeingNF-κBcomplexestotranslocatetothenucleus,bindtoNF-κBDNAresponseelements,andinducethetranscriptionofthetargetgenes.
TheNF-κBTransientPackisdesignedtoprovidethetoolsnecessaryfortransientlytransfectingandmonitoringtheactivityoftheNF-κBsignalingpathwayinculturedHEK293cells.Thekitcontainstransfection-readyvectorscontainingfireflyluciferaseasaNF-κBpathway-responsivereporterandconstitutivelyexpressingRenillaluciferaseasatransfectioncontrol. ItalsoincludestheDualLuciferasedetectionreagentstodetectbothluciferaseactivitiesandspecializedmediumforgrowingandassayingHEK293cells.
ThekeytotheNF-κBTransientPackistheNF-κBluciferasereportervector.ThisreportercontainsafireflyluciferasegeneunderthecontrolofmultimerizedNF-κBresponsiveelementlocatedupstreamofaminimalpromoter.TheNF-κBreporterispremixedwithconstitutively-expressingRenillaluciferasevector,whichservesasaninternalcontrolfortransfectionefficiency.
Thepackalsoincludesanon-inducIBLefireflyluciferasevectorpremixedwithconstitutively-expressingRenillaluciferasevectorasanegativecontrol.Thenon-inducibleluciferasevectorcontainsafireflyluciferasegeneunderthecontrolofaminimalpromoter,withoutanyadditionalresponseelements.Thenegativecontroliscriticaltodeterminingpathwayspecificeffectsandbackgroundluciferaseactivity.
Additionally,thepackincludescellculturemedium(BPSMedium1)thathasbeenoptimizedforusewithHEK293cells*.BPSMedium1includesMEMmedium,10%fetalbovineserum,1%non-essentialaminoacids,sodiumpyruvate,and1%Pen/Strep. Finally,thepackprovidestheDualLuciferase(Firefly-Renilla)AssaySystem.Thesereagentsprovidehighlysensitive,stabledetectionoffireflyluciferaseactivityandRenillaluciferaseactivity. Thedualluciferasereagentscanbeuseddirectlyincellsingrowthmedium,andcanbedetectedwithanyluminometer;automatedinjectorsarenotrequired.
*Note: thekitmaybeusedwithothercelllinesthanHEK293,butanalternatecellculturemediummayberequiredforoptimalcellgrowth.
•ScreenactivatorsorinhibitorsofNF-κBsignalingpathway.
•StudyeffectsofRNAiorgeneoverexpressionontheactivityoftheNF-κBpathway.
2.Baeuerle,P.A.(1998).Pro-inflammatorysignaling:lastpiecesintheNF-kappaBpuzzle?CurrBiol.8(1):R19-R22.
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