2.12. Western blotting

The left ventricular tissue of hearts and H9c2 cells were harvested after diverse treatments and prepared for western blotting. The separation of cytosol and mitochondrial protein fraction from cardiac tissues and H9c2 cells were obtained by using a mitochondria isolation kit (Beyotime, Shanghai, China) according to the manufacture's instruction. After separating the protein samples by SDS‐PAGE, the proteins were transferred onto a PVDF membrane (Millipore, Billerica, MA, USA) and blocked with 5% nonfat milk in TBST. The membrane was then incubated with primary antibodies at 4°C overnight. The antibodies against Bip/GRP78 (1: 1,000), IRE1α (1: 1,000), Total OXPHOS, irisin, and p‐ IRE1α (1: 1,000) were purchased from Abcam while the antibodies against XBP‐1s (1:1000), ATF4 (1:1000), CHOP (1:1000), Caspase 3 (1:1000), Bax (1:1000), Bcl‐2 (1:1000), Cyto C (1:1000), and GAPDH (1:5000) were purchased from Cell Signaling Technology. After wash with TBST, the membranes were incubated with HRP‐conjugated second antibodies for 1.5‐2 h at RT. Then the proteins were visualized using chemiluminescent reagents (Millipore, Billerica, MA, USA) under ChemiDoc Imaging System (Bio‐Rad Laboratories, Hercules, CA, USA) and the densities of the bands were quantified by Image Lab software (Bio‐Rad Laboratories, Hercules, CA, USA). GAPDH was used as an internal reference.