Mitochondrial isolation buffer

Fresh myocardium tissues were homogenized with a glass homogenizer on ice within 1h after euthanasia. Isolation buffer (c3606, Beyotime, Shanghai, China) included in the tissue mitochondria isolation kit was used to isolate mitochondria from cardiac tissue. According to the manufacturer’s instructions for the kit, isolation buffer was added to homogenized tissue after the cardiac tissue was dissected and washed with ice-cold PBS solution and trypsin solution. Then, the supernatant was collected after the homogenate was centrifuged (600×g/min for 5min, 4°C) in a low-temperature centrifuge. After the supernatant was further centrifuged (11,000×g/min for 10min, 4°C), the mitochondria (sediment) was isolated. The mitochondrial pellet was resuspended in mitochondrial isolation buffer (Beyotime, Shanghai, China) as previously described (Ouyang et al., 2016 (link)).

Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) analysis was performed as described previously^{13 (link)}. In brief, harvested cells resuspended in mitochondrial isolation buffer (Beyotime Biotechnology) were subjected to dounce-homogenization to lyse the cell. The homogenate was centrifuged at 700 × g for 10 min at 4 °C to spin out the cell debris and nucleus. The supernatant was further centrifuged at 10,000 × g for 30 min at 4 °C to pellet the intact crude mitochondria. Crude mitochondria (P5) were lysed in 1×sample buffer (0.5× TBE, 10% glycerol, 2% SDS and 0.0025% bromophenol blue) and loaded onto a vertical 1.5% agarose gel (Bio-Rad). The proteins were transferred to an Immobilon membrane (Millipore) as mentioned above for further immunoblot analysis after electrophoresis in the running buffer (0.5× TBE and 0.1% SDS) for 35 min with a constant voltage of 100 V at 4 °C.

Fresh gastrocnemius muscle tissues were homogenized by a glass homogenizer on ice for 1 h after sacrifice according to the guidelines of the tissue mitochondria isolation kit (Beyotime, China, c3606). After the homogenate was centrifuged at 600 g/min for 10 min, the divided supernatant was further centrifuged at 11000 g/min for 15 min. The mitochondrial pellet was resuspended in mitochondrial isolation buffer (Beyotime, Shanghai, China).