Fluorescence resonance energy transfer analysis for detecting protein kinase C activation

Objective To establish a sensitive and direct method for detecting the activation of protein kinase C(PKC) using fluorescence resonance energy transfer(FRET) technique.Methods HEK293 cells were transfected with C kinase activity reporter(CKAR) plasmid or/and parathyroid receptor 1 plasmid,and after incubation for 72 h,the fluorescence resonance energy transfer was measured with or without parathyroid or TPA stimulation.Results TPA reduced the efficiency of FRET and increased the emission ratio of CFP/YFP(C/Y) in HEK293 cells transfected with CKAR.PTH(1-34) could increase the emission ratio of C/Y in HEK293 cells co-transfected with CKAR and PTHR1 but not in cells transfected with CKAR.Conclusion FRET analysis using CKAR can be utilized to detect the activation of PKC,which provides a useful means for studying the signaling pathways associated with PKC.