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.
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.