Western Blot Assay

Total protein from the heart tissue specimens or the cultured cells was extracted by RIPA lysis buffer. Highly enriched fractions of cytoplasmic and nuclear proteins were extracted using NE‐PER^™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833). The protein concentrations were determined using a BCA Protein Assay Kit (Pierce, 23225). The protein extracts were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore). After blocking with 5% fat‐free milk, the membranes were incubated with the indicated primary antibodies on a rocking platform overnight at 4°C. The membranes were then incubated with the corresponding peroxidase‐conjugated secondary antibodies for 1 hour at room temperature. The immunoreactive bands were visualized using an enhanced chemiluminescence detection kit (Bio‐Rad, 170‐5061), and the signals were detected and quantified using a Bio‐Rad ChemiDoc^™ XRS+System (Bio‐Rad). Specific protein expression levels were normalized to GAPDH for total cell and cytoplasmic lysates or to Lamin B for the nuclear proteins.