Cells and heart tissues were lysed with radioimmunoprecipitation assay lysis buffer (Solarbio, Beijing, China). Equal amounts of protein (50 to 60 μg) were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to polyvinylidene difluoride membranes, and incubated with primary antibodies as indicated for each experiment, followed by incubation with horseradish peroxidase–conjugated secondary antibodies (1:2500) for 1 hour at 20° to 23°C.All blots were developed using a chemiluminescent system, and signal intensities were analyzed with a Gel-Pro 4.5 Analyzer (Media Cybernetics, Rockville, MD, USA).