Primary cardiomyocytes or heart tissues were lysed with radioimmunoprecipitation assay lysis buffer (Solarbio). 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 in each experiment, and then with horseradish peroxidase–conjugated secondary antibodies (1:2500), as described previously (6, 45). Western blot signal intensities were analyzed with a Gel-Pro 4.5 analyzer (Media Cybernetics, USA) and were normalized to controls.

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