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Signosis/NRF2/ARE Luciferase Reporter NIH3T3 Stable Cell Line/SL-0047-NP/1 Ea
Description:
NRF2/AREResponsiveLuciferaseReporterNIH3T3StableCellLineisderivedfromMousefibroblast,andstablyexpressfireflyluciferasereportergeneunderthecontroloftheNRF2/AREresponseelement. ThiscelllineisanidealcellularmodelformonitoringtheactivationofAntioxidantResponsePathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
NRF2playsacrucialroleincellularanti-oxidantdefense,makingitatherapeutictargetforneurodegenerativediseasesandcancer. Undernormalconditions,NRF2localizesinthecytosolandisrapidlydegradedbytheproteasome. Underoxidativestress,NRF2isstABIlizedandtranslocatestothenucleuswhereitbindstoaDNApromoterandinitiatesgeneexpression. Inthenucleus,NRF2formsaheterodimerwithasmallMafproteinandbindstotheAntioxidantResponseElementintheupstreampromoterregionofmanyantioxidativegenes,andinitiatestheirtranscription.
ThisNRF2luciferasereporterstablecelllinehasbeenstablytransfectedwithpTA-ARE-luciferasereportervector,whichcontains4repeatsofantioxidantresponsebindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion,alongwitha hygromycinexpressionvector. Followingselection,the hygromycinresistantclonesweresubsequentlyscreenedforTBHQ-inducedluciferaseactivity.Theclonewiththehighestfoldinductionwasselectedandexpandedtoproducethisstablecellline.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisoftheNRF2PathwayReporterNIH3T3StableCellLineinresponsetostimuli. Thecellswereseededona96-wellplatefor8hoursorovernightwithDMEMincluding10%FBS.Thecellsthenweretreatedwithorwithout10μMTBHQor10μM4HNE respectivelyinDMEMand0.1%FBSfor16hours.