TheNotchsignalingpathwaycontrolscellfatedecisionsinvertebrateandinvertebratetissues.NOTCHsignalingistriggeredthroughthebindingofatransmembraneligandtoNotchtransmembranereceptor(NOTCH1/NOTCH2/NOTCH3/NOTCH4)onaneighboringcell.ThisresultsinproteolyticcleavageoftheNOTCHreceptor,releasingtheconstitutivelyactiveintracellulardomainofNOTCH(NICD).NICDtranslocatestothenucleusandassociateswithtranscriptionfactorsCSL(CBF1/RBPJκ/SuppressorofHairless/Lag-1)andcoactivatorMastermindtoturnontranscriptionofNotch-responsivegenes.
TheNotch1/CSLTransientPackisdesignedtoprovidethetoolsnecessaryfortransientlytransfectingandmonitoringtheactivityoftheNotchsignalingpathwayinHEK293culturedcells.Thekitcontainstransfection-readyvectorscontainingfireflyluciferaseasaNotchpathway-responsivereporterandconstitutivelyexpressingRenillaluciferaseasatransfectioncontrol. ItalsoincludestheDualLuciferasedetectionreagentstodetectbothluciferaseactivitiesandspecializedmediumforgrowingandassayingHEK293cells.
ThekeytotheNotch1/CSLTransientPackistheexpressionvectorforNOTCH1thathasadeletionoftheentireextracellulardomain(Notch1DE).Insidethecells,theNOTCH1DEcanbecleavedbyγ-secretaseandactiveNOTCH1NICDisreleasedintothenucleus.ThekitalsocontainsCSL(CBF1/RBP-Jk)luciferasereportervector,whichisaNotchpathway-responsivereporter.ThisreportercontainsthefireflyluciferasegeneunderthecontrolofmultimerizedCSLresponsiveelementsupstreamofaminimalpromoter.TheCSL(CBF1/RBP-Jk)reporterispremixedwithconstitutivelyexpressingRenilla(seapansy)luciferasevector,whichservesasaninternalpositivecontrolfortransfectionefficiency.
Thepackalsoincludesanon-inducIBLefireflyluciferasevectorpremixedwithconstitutively-expressingRenillaluciferasevectorasanegativecontrol.Thenon-inducibleluciferasevectorcontainsafireflyluciferasegeneunderthecontrolofaminimalpromoter,butwithoutanyadditionalresponseelements.Thenegativecontroliscriticalfordeterminingpathwayspecificeffectsandbackgroundluciferaseactivity.
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,
•ScreenactivatorsorinhibitorsofNotchsignalingpathway
•StudyeffectsofRNAiorgeneoverexpressionontheactivityofNotchpathway.
2.Kanungo,etal.(2008)TheNotchsignalinginhibitorDAPTdown-regulatescdk5activityandmodulatesthedistributionofneuronalcytoskeletalproteins.J.NeuRochem.106:2236.
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