HL-1 cardiomyocytes or human tissue samples were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Cell Signaling) supplemented with protease and phosphatase inhibitors (complete Mini and PhosSTOP; Roche) on ice for 5-15 min. After centrifugation to pellet nondissolved material, the protein concentration of each sample was determined using the bicinchoninic acid method (BCA; Thermo Fisher).
Equal amounts of protein homogenates were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Membranes were blocked in blocking buffer and probed with the primary antibody (overnight at 4°C). The following primary antibodies were used: RYR2 (2446725; Millipore), p21 (ab109199; ABCAm), p53 (2524; Cell Signaling), p16 (ab211542; ABCAm), γH2AX (9718; Cell Signaling), CACANA1C (Cav1.2; ab84814; ABCAm), SERCA2 (ab150435; ABCAm), and PLB (ab219626; ABCAm). The membranes were subsequently incubated with anti-mouse or anti-rabbit secondary antibodies (ABCAm). Signals were detected using the enhanced chemiluminescence (ECL) detection method (Thermo Fisher) and quantified by densitometry (ImageJ). The original uncropped blots are available in the Supplementary Information section.
Equal amounts of protein homogenates were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Membranes were blocked in blocking buffer and probed with the primary antibody (overnight at 4°C). The following primary antibodies were used: RYR2 (2446725; Millipore), p21 (ab109199; ABCAm), p53 (2524; Cell Signaling), p16 (ab211542; ABCAm), γH2AX (9718; Cell Signaling), CACANA1C (Cav1.2; ab84814; ABCAm), SERCA2 (ab150435; ABCAm), and PLB (ab219626; ABCAm). The membranes were subsequently incubated with anti-mouse or anti-rabbit secondary antibodies (ABCAm). Signals were detected using the enhanced chemiluminescence (ECL) detection method (Thermo Fisher) and quantified by densitometry (ImageJ). The original uncropped blots are available in the Supplementary Information section.