4.8. Cytosol/Mitochondria Fractionation

Mitochondrial fractionation was performed as previously described [21]. Briefly, the cells were collected after trypsinization and washed once with PBS. Collected cells were lysed in extraction buffer containing protease. After incubating for 10 min on ice, lysates were centrifuged at 700× g for 10 min at 4 °C. Collected supernatants were centrifuged at 10,000× g for 30 min at 4 °C, and the cytosolic fraction (supernatants) was collected. The mitochondrial fraction (pellets) was resuspended in the extraction buffer, and the protein amount of each fraction was measured by a bicinchoninic acid protein assay (BCA Protein Assay Kit; Thermo Fisher Scientific, Rockford, IL, USA).