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[Fluorescence resonance energy transfer analysis for detecting protein kinase C activation]
Jun-qing Li1, Jian-ming Jiang, Rui-qiang Feng
1Department of Spinal Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. ljqiskobebryant@163.com
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.
