Quantitative Western Blot Analysis of Mitochondrial Proteins

Western blot assays were performed as described previously^{28 (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 minutes in laemmli sample buffer (Bio-Rad), but other proteins were heated at 95°C for 5 minutes 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.