Mitochondrial Protein Western Blot Analysis

Western blot assays were performed as described previously^{13 (link)}. In brief, cells or purified mitochondria were resuspended in RIPA buffer (TEKnova) containing 1% protease inhibitor cocktail (Sigma). Protein concentration was determined using BCA protein assay (Pierce). To analyze mitochondrial proteins, lysates were heated at 42 °C for 5 min in Laemmli sample buffer (Bio-Rad), but proteins from whole cell lysate were heated at 95 °C for 5 min in the same buffer. 10–30 µg protein lysates were loaded onto 4–20% gradient SDS PAGE (Bio-Rad) and transferred to PVDF membranes (Roche). Membranes were probed with the primary antibodies followed by washing and adding the fluorescent secondary antibodies. Antibody-antigen reaction profiles were visualized and quantified by Odyssey XF Imager (LI-COR). Antibodies are summarized in Supplementary Table 3.