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1、protocolUsingpatternedsupportedlipidmembranestoinvestigatetheroleofreceptororganizationinintercellularsignalingPradeepMNair1–3,KhalidSalaita4,5,RebeccaSPetit1–3&JayTGroves1–3,61DepartmentofChemistry,HowardHughesMedicalInstitute,UniversityofCalifornia,Berkeley,California,USA.2LawrenceBerkeley
2、NationalLaboratory,PhysicalBiosciencesDivision,Berkeley,California,USA.3LawrenceBerkeleyNationalLaboratory,MaterialsSciencesDivision,Berkeley,California,USA.4DepartmentofChemistry,EmoryUniversity,Atlanta,Georgia,USA.5WinshipCancerInstitute,EmoryUniversity,Atlanta,Georgia,USA.6ResearchCentero
3、fExcellenceinMechanobiology,NationalUniversityofSingapore,Singapore,Singapore.CorrespondenceshouldbeaddressedtoJ.T.G.(jtgroves@lbl.gov).Publishedonline31March2011;doi:10.1038/nprot.2011.302physicalinputs,bothinternalandexternaltoacell,candirectlyalterthespatialorganizationofcellsurfacerecept
4、orsandtheirassociatedfunctions.Herewedescribeaprotocolthatcombinessolid-statenanolithographyandsupportedlipidmembranetechniquestotriggerandmanipulatespecificreceptorsonthesurfaceoflivingcellsandtodevelopanunderstandingoftheinterplaybetweenspatialorganizationandreceptorfunction.Whileexistingp
5、rotein-patterningtechniquesarecapableofpresentingcellswithwell-definedclustersofprotein,thisprotocoluniquelyallowsforthecontrolofthespatialorganizationoflaterallyfluidreceptor-ligandcomplexatanintermembranejunction.acombinationofimmunofluorescenceandsingle-cellmicroscopymethodsandcomplementa
6、rybiochemicalanalysesareusedtocharacterizereceptorsignalingpathwaysandcellfunctions.theprotocolrequires2–5dtocompletedependingontheparameterstobestudied.Inprinciple,thisprotocoliswidelyapplicabletoeukaryoticcellsandhereinisspecificallydevelopedtostudytheroleofphysicalorganizationandtransloca
7、tionoftheepha2receptortyrosinekinaseacrossalibraryofmodelbreastcancercelllines.Allrightsreserved..IncIntroDuctIonJuxtacrinesignaling,inwhichreceptorsandtheircognateligandsandalternativelithographictechniques,whicharesimplerandareexpressedonapposedc
