Mitochondrial and Cytosolic Fractionation

Mitochondrial and cytosolic protein fractions were prepared using a Cell Mitochondria Isolation Kit according to a standard procedure (Beyotime Institute of Biotechnology, China). Briefly, 5 × 10⁷ cells were harvested and washed with ice-cold PBS. The cells were resuspended in mitochondrial isolation reagent containing phenylmethylsulfonyl fluoride and protease inhibitors and homogenized in an ice-cold grinder. The homogenates were centrifuged at 600 ×g for 10 min and then the supernatants were further centrifuged at 11,000 ×g for 10 min at 4°C. The supernatants were decanted as the cytosolic fraction and the pellets were resuspended in 100 μl mitochondrial lysis buffer, which was kept as the mitochondrial fraction. The mitochondrial and cytosolic protein were subjected to western blotting. Tom20 served as a mitochondrial marker and α-GAPDH served as a cytosolic marker.