Ab87399

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).

Livers were placed into lysis buffer (10 mM Tris–HCl [pH 8.0], 10 mM NaCl, and 0.5% NP-40) containing protease inhibitors (Roche) and homogenized by a shaker. Then, lysates were centrifuged at 12,000 g for 20 min at 4 °C. The supernatants containing loading buffer were boiled for 5 min. Western blotting transfers were carried out in BioRad transblot chambers. After blocking in 5% milk in phosphate-buffered saline Triton X100 (PBST) for 1 h, the membranes were incubated at 4 °C overnight with anti-NDUFS4 mouse antibody (Abcam, ab87399) followed by incubation with an HRP-conjugated secondary antibody for 1 h at room temperature. The signals were visualized with the Super Signal West Femto reagent (Thermo scientific).

Blood inside organs were removed by rinsing tissues with PBS. Tissues were snap-frozen under liquid nitrogen. Tissues were homogenized in RIPA buffer (Sigma) with protease (Roche), deacetylase (nicotinamide, Tricostatin A) and phosphatase inhibitors (Roche)^{7 (link)}. Protein concentrations of samples were determined by Lowry assay and each amount of protein (30–50 ug per sample) were loaded for SDS-PAGE. Antibodies from the following companies were used for Western blot analysis: Phospho-H2Ax (1:500, NBP1-19255-Novus Biological), PAR (1:500, AM80100UG-Millipore), actin (1:5000, sc-8432-Santa Cruz), nitrotyrosine (1:500, 06-284-Millipore) acetyl-lysine (1:1000, 9441-Cell signaling), HIF1a (1:400, 14179-Cell Signaling), GLUT1 (1:500, ab652-Abcam), PDK4 (1:1000, 3820-Cell Signaling), LDHA (1:1000, 3582-Cell Signaling), SDHA (1:10000, ab14715-Abcam), Ndufs4 (1:1000, ab87399-Abcam,), SOD2 (1:1000, ab16956-Abcam) SOD2-Ac (1:1000, ab137037-Abcam), GDH (1:1000, 12793-Cell Signaling). Blots were blocked in 5% BSA-TBST. Antibodies were diluted in 5% BSA-TBST. Protein bands were visualized with chemiluminescence assay (Pierce) with secondary antibodies coupled with HRP. The protein abundance was analyzed by densitometry with ImageJ. SDHA or actin were used as a loading control for quantification.