Mitochondrial Fractionation and Localization

Mitochondrial fractionation was performed using the Mitochondrial Isolation Kit for Cultured Cells (Thermo Fisher Scientific, MA, USA) following the manufacturer’s instructions. In brief, 1 × 10⁷ cells were collected and incubated in mitochondria isolation buffers on ice for 5 min. After homogenization, nuclei and intact cells were discarded after centrifugation at 700 g for 10 min at 4°C. The supernatant enriched with the cytosolic fraction and the pellet enriched with the mitochondrial fraction were obtained after centrifugation at 12,000 g for 15 min at 4°C. For mitochondrial protein-localization assays as previously described [25 (link)], the purified mitochondria were divided into three groups. The first group was not treated, as a control. The second group was treated with 3 μM proteinase K (Invitrogen, MA, USA). The third group was treated with 3 μM proteinase K and 0.1% Triton X-100 solution. Three groups of samples were placed in a 37°C water bath for 30 min. All samples were prepared for further immunoblotting.