Sc 135900

SDS-PAGE and immunoblotting were performed as previously described (Maxfield et al., 2015 (link)). Antibodies used for immunoblotting were as follows: COX6B2 (1:1000, SAB1401983, MilliporeSigma), COX6B1 (1:1000, sc-393233, Santa Cruz Biotechnology), COXIV (1:5000, 4850, Cell Signaling Technology), β-Actin (1:10,000, sc-47778, Santa Cruz Biotechnology), V5 (1:5000, R960-25, Thermo Fisher Scientific), HIF-1α (1:1000, 610959, BD Biosciences), Cleaved Caspase-3 (1:500, 9661, Cell Signaling Technology), PARP (1:1000, 9532, Cell Signaling Technology), ERK (1:3000, sc-135900, Santa Cruz Biotechnology), NDUFA9 (1:1000, 459100, Thermo Fisher Scientific), UQCRC2 (1:1000, ab14745, Abcam). Proteins were quantitated by ImageJ software and normalized to CTRL groups. Incorporated COX6B1, COX6B2 or COXIV in individual complex (monomers (IV) and dimers (IV₂) and supercomplexes (III₂+IV and I+III₂+IV_n)) was normalized to total complexes (sum of IV, IV₂, III₂+IV and I+III₂+IV_n).

The experiments were performed as described previously [44 (link)]. The protein concentrations of cell and tissue lysates were measured using the Lowry assay, and 30 µg of total protein were separated by SDS-PAGE in 7.5, 10, or 12.5% gels depending on the molecular weight of the target. Primary antibodies against HMGB1 (1:1000; 66525-1-Ig, Proteintech, Rosemont, IL, USA), pDRP1(Ser616) (1:500; AP0849, ABclonal, MA, USA), pERK (1:500, #9101, Cell Signaling, Beverly, MA, USA), ERK (1:500; sc-135900, Santa Cruz Biotechnology, CA, USA), α-tubulin (1:2000; T5168, Sigma, Louis, MO, USA), Lamin A/C (1:1000, GTX101127, GeneTex, Irvine, CA, USA), and α-actin (1:2000, sc-137179, Santa Cruz Biotechnology) were used.