β actin

Isolated cardiomyocytes or dissected mouse ventricular heart tissue samples were lysed in ice‐cold radioimmunoprecipitation assay buffer (Dingguo Changsheng, Beijing, China) with protease and phosphatase inhibitors. Protein concentrations were determined with the BCA Protein Quantitative Analysis kit (Fudebio‐tech, Hangzhou, China). Protein samples were separated by 8% to 12% SDS‐PAGE and transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were incubated at room temperature for 2 hours in blocking buffer (5% BSA in Tris‐buffered saline and Tween 20 buffer). After blocking, the membranes were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: Sirt1 (Cell Signaling Technology, MA) and β‐actin (Biosynthesis, Beijing, China). After the membranes were washed 3 times with Tris‐buffered saline and Tween 20, they were incubated with a donkey anti‐rabbit IgG horseradish peroxidase (Abcam) for 1 hour at room temperature. The membranes were developed using the enhanced chemiluminescence method, according to the manufacturer's instructions (Millipore), and detected on the chemiluminescence imager GeneGnome XRQ (Syngene, MD). To calculate the relative density, ImageJ software was used and the intensity of each protein was normalized to that of β‐actin.