Anti cleaved caspase 3

Protein from the heart and cardiomyocytes was extracted in the lysis buffer with protease inhibitors. The protein concentrations were determined using Pierce BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Equivalent levels of proteins were denatured and resolved with 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and then transferred to nitrocellulose membranes, incubated with 5% skimmed milk, and probed with primary antibodies overnight at 4°C. Primary antibodies were diluted as follows: anti‐cleaved‐caspase‐3 (1:1,000; Signalway Antibody LLC), anti‐mouse GAPDH, anti‐HA‐tag, anti‐m‐calpain, anti‐μ‐calpain, anti‐caspase‐12, anti‐IRE1 (1:1,000; Cell Signaling Technology), anti‐calpain‐7 (1:1,000; ProteinTech Group, Chicago, IL, USA), anti‐P‐IRE1(1:1,000; Littleton, CO, USA), anti‐ATF6 (1:500, Santa Cruz, CA, USA), anti‐LC3A/B, anti‐phospho‐MLKL, anti‐MLKL (1:1,000; Cell Signaling Technology), anti‐caspase‐4, anti‐caspase‐8, and anti‐caspase‐9 (1:1,000; ABclonal). The membranes were washed and then incubated in horseradish peroxidase‐labeled secondary antibody for 1–2 h at room temperature. Proteins were detected using enhanced chemiluminescence reagents (Thermo Scientific, Waltham, MA, USA), and blots were quantified with ImageJ and normalized by GAPDH.

Anti-Mcl-1 (1:1,000), anti-BAD (1:500), anti-Bcl-2 (1:1,000), anti-Bcl-XL (1:500), anti-cleaved caspase-3 (1:500), anti-Stat3 (1:1,000), anti-LC3B (1:1,500), anti-beclin1 (1:1,000), and anti-β-actin (1:1,000) antibodies were purchased from Signalway antibody (College Park, Maryland, USA). Anti-Akt (1:1,000), anti-p-Akt (1:1,000), anti-p- anti-mTOR (1:1,000), mTOR (1:1,000) antibodies were bought from Cell Signaling Technology Inc. (Danvers, MA, USA). Anti-P62 (1:1,000) was purchased from Proteintech Group Inc. (Wuhan, China) S1P was bought from Sigma-Aldrich (Cat. S9666, St. Louis, MO, USA). FTY720 was bought from Cayman Chemical Company (Ann Arbor, MI, USA).