Mitochondrial isolation

Mitochondria were isolated as previously published (Chen et al, 2017). In brief, MEFs were infected with retroviral particles containing pMXs‐3XHA‐EGFP‐OMP25, selected with 10 μg/ml Blasticidin and the expression of HA‐tag was verified by SDS–PAGE. The day of experiment, ~ 30 million MEFs were collected, washed with KPBS buffer (136 mM KCl and 10 mM KH₂PO₄, pH 7.25), and homogenized with 25 stokes of the plunger at 1000 rpm at 4°C. Nuclei and debris were discarded by centrifugation at 1,000 g for 2 min at 4°C. The supernatant was collected and subjected to immunocapture with prewashed anti‐HA magnetic beads for 30 min on end‐over‐end rotator 4°C. The beads were then washed three times and resuspended in 500 μl KPBS. 30% of the suspension beads was set aside and used for immunoblotting. The remaining beads were store at −150°C for the indicated analysis.