Ab14745

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).

Skeletal muscle and fibroblast homogenates were obtained according to previously described methodologies.³⁰ (link) 30–40 μg (S1–S3) and 20 μg (S4) of whole-cell protein extracts were separated by SDS polyacrylamide (12%) electrophoresis and then wet transferred to polyvinyl difluoride (PVDF) membranes. For S4, a 4%–12% gradient gel was used. Immunological detection of proteins was carried out with the following primary antibodies: C1QBP (ab24733, Abcam), β-actin (A1978, Sigma), α-tubulin (ab7291, Abcam), and OXPHOS complex-specific antibodies (NDUFS3 [ab14711, Abcam], NDUFB8 [ab110242, Abcam], NDUFA9 [MS111, Molecular Probes], SDHA [459200, MitoSciences], SDHB [ab14714, Abcam], UQCRC2 [ab14745, Abcam], COXI [ab14705, Abcam], COXII [ab110258, Abcam], COXIV [ab14744, Abcam], and ATP5A [ab14748, Abcam]). Species-appropriate horseradish-peroxidase-conjugated secondary antibodies (DAKO, P0399, and P0260) were used.

Total proteins were extracted using radioimmunoprecipitation assay buffer. Protein concentration was assessed with a Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were separated by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. The same amount of protein (μg) was loaded in each lane. After electrophoresis, the proteins were transferred to a polyvinylidene difluoride or nitrocellulose membrane and subsequently subjected to immunoblotting analysis using appropriate antibodies. The amount of loading was further determined using a western blotting housekeeping protein (lamin A, 1:5000 dilution). Using a horseradish peroxidase-conjugated secondary antibody, protein bands on the blots were visualized with enhanced chemiluminescent western blot detection reagent. Antibodies including anti-tubulin (Abcam, ab7291), anti-LDHB (Abcam, ab75167), anti-complex I (Abcam, ab110242), anti-complex II (Abcam, ab14714), anti-complex III (Abcam, ab14745), anti-complex IV (Abcam, ab14705), anti-complex V (Abcam, ab14748) were purchased from commercial company (Abcam, Cambridge, UK).