Mitochondrial Protein Complex Analysis

Mitochondrial pellets were solubilized with digitonin at 4:1 g protein and fractionated in blue native (BN) gels [28 (link)]. A total of 30 μg of mitochondrial proteins were fractionated in NativePAGE 3–12% Bis-Tris Protein Gels, 1.0 mm, 10-well (Invitrogen) at 4°C using cathode (50 mM Tricine, 15 mM Bis-Tris, pH 7.0 and 0.02% Serva blue G) and anode buffers (50 mM Bis-Tris, pH 7.0). Electrophoresis was carried out at a constant voltage (70 V) until the samples entered the polyacrylamide gradient (approximately 30 minutes) and then at a constant current (15 mA) until the dye reached the end of the gel (approximately 1 hour). After fractionation, the gels were electroblotted onto PVDF membranes and processed for immunoblot analysis as described above. The following primary antibodies were used: mouse anti-NADHs9 (NDUFA9, clone 15/22-5 [89 (link)], 1:1,000), mouse anti-NDUFS3 (clone 17D95, Abcam, ab14711, 1:1,000), mouse anti-NDUFS4 (clone 2C7CD4AG3, Abcam, ab87399, 1:1,000), mouse anti-SDH-A (clone 2E3GC12FB2AE2, Abcam, ab14715, 1:1,000), mouse anti-UQCRC2 (clone 13G12AF12BB11, Abcam, ab14745, 1:1,000), mouse anti-MT-CO1 (clone 1D6E1A8, Invitrogen, #459600, 1:1,000), mouse anti-MT-CO3 (clone DA5BC4, Abcam, ab110259, 1:1,000), mouse anti-COX IV (clone 20E8C12, Abcam, ab14744, 1:1,000), and rabbit anti-β-F1 [93 (link)] (1:20,000).