Thetransforminggrowthfactorbeta(TGFβ)signalingpathwayisinvolvedinadiverserangeofcellprocessessuchasdifferentiation,cellcyclearrest,andimmuneregulation.TGFβsignalinghasbeenlinkedtocardiacdisease,cancer,Alzheimer’sandotherhumandiseases.TGFβproteinsbindtoreceptorsonthecellsurface,initiatingasignalingcascadethatleadstophosphorylationandactivationofSMAD2andSMAD3,whichthenformacomplexwithSMAD4.TheSMADcomplexthentranslocatestothenucleusandbindstotheSMADbindingelement(SBE)inthenucleus,leADIngtotranscriptionandexpressionofTGFβ/SMADresponsivegenes.
TheSBETransientPackisdesignedtoprovidethetoolsnecessaryfortransientlytransfectingandmonitoringtheactivityofTGFβ/SMADsignalingpathwayintheculturedcells.Thekitcontainstransfection-readyvectorscontainingfireflyluciferaseasaTGFβ/SMADpathway-responsivereporterandconstitutivelyexpressingRenillaluciferaseasatransfectioncontrol. ItalsoincludestheDualLuciferasedetectionreagentstodetectbothluciferaseactivitiesandspecializedmediumforgrowingandassayingHEK293cells.
ThekeytotheSBETransientPackistheSBEluciferasereportervector,whichisaTGFβpathway-responsivereporter.ThisreportercontainsafireflyluciferasegeneunderthecontrolofmultimerizedSBEresponsiveelementlocatedupstreamofaminimalpromoter.TheSBEreporterispremixedwithconstitutivelyexpressingRenillaluciferasevectorthatservesasinternalcontrolfortransfectionefficiency.
Thepackalsoincludesanon-inducIBLefireflyluciferasevectorpremixedwithconstitutivelyexpressingRenillaluciferasevectorasnegativecontrol.Thenon-inducibleluciferasevectorcontainsafireflyluciferasegeneunderthecontrolofaminimalpromoter,withoutanyadditionalresponseelements.Thenegativecontroliscriticaltodeterminingpathway-specificeffectsandbackgroundluciferaseactivity.
Additionally,thepackincludescellculturemedium(BPSMedium1)thathasbeenoptimizedforusewithHEK293cells*.BPSMedium1includesMEMmedium,10%fetalbovineserum,1%non-essentialaminoacids,sodiumpyruvate,and1%Pen/Strep. Finally,thepackprovidestheDualLuciferase(Firefly-Renilla)AssaySystem.Theseluciferasereagentsprovidehighlysensitive,stabledetectionoffireflyluciferaseactivityandRenillaluciferaseactivity. Thedualluciferasereagentscanbeuseddirectlyincellsingrowthmedium,andcanbedetectedwithanyluminometer;automatedinjectorsarenotrequired.
*Note: thekitmaybeusedwithothercelllinesthanHEK293,butanalternatecellculturemediummayberequiredforoptimalcellgrowth.
•ScreenactivatorsorinhibitorsofTGFβ/SMADsignalingpathway.
•StudyeffectsofRNAiorgeneoverexpressionontheactivityofTGFβpathway.
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