Ab119686

Immunoblot analysis was used to assess protein levels of subunits of complexes I, III and IV, and subunits of F₀F₁-ATPase in renal cortical mitochondria using 20 μg of mitochondrial protein, as described previously [57 (link),61 (link)]. Antibodies against NDUFA9, NDUFS3, and NDUFB7 subunits of complex I (Cat #A21344, A21343, and 21359) were purchased from Molecular Probes/ThermoFisher Scientific (Waltham, MA, USA). Antibodies against α- and γ-subunits of F₀F₁-ATPase (Cat #ab14748 and #ab119686, respectively) and the core protein 1 (UQCRC1), core protein 2 (UQCRC2), and Rieske protein subunits of complex III (Cat #ab110252, #ab14745, and #ab14746, respectively) were supplied by Abcam (Cambridge, MA, USA). Antibodies against β-subunit of F₀F₁-ATPase (Cat #A21351) were supplied by Life Technologies Corporation (Carlsbad, CA, USA). Cytochrome oxidase subunit I antibody was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA, Cat #sc-58347). Citrate synthase levels served as the loading control using the citrate synthase antibody (Cat #D7V8B) supplied by Cell Signaling Technology (Danvers, MA, USA).

Blue Native gel electrophoresis (PAGE) was performed by isolating mitochondrial proteins from mutant and control cybrid cell lines as described elsewhere [21 (link)]. Samples containing 30 μg of proteins were separated on 3~12% Native PAGE gel. The primary antibodies applied for this experiment were NDUFA9 (Abcam, ab128744), SDHA (Abcam, ab14715), UQCRC2 (Abcam, ab203832), COX5A (Abcam, ab110262), and ATP5C (Abcam, ab119686). As a secondary detection antibody, an ECL system was used for band detection, after which densitometric calculations were performed as shown in Western blotting.