Immunoblot Analysis of Mitochondrial OXPHOS

Preparation of tissue lysate was carried out as described previously [93 (link)]. From this, 10 μg protein was loaded into 4–20% gradient gels (Mini-PROTEAN® TGX™ Precast Protein Gels, 15-well, 15 μl #4561096, BioRAD) alongside a protein ladder (Precision plus protein dual color standards #1610374, BioRAD). The gel was transferred to nitrocellulose membrane, and this was stained in Ponceau S. The membrane was blocked in 5% Skimmed milk-TBS-T for 1 h prior to primary antibody incubation (Total OXPHOS antibody cocktail, ab110412, Abcam, RRID:AB_2847807; 1:500 in 1% Milk TBS-T) overnight at 4 °C. The membrane was incubated with secondary antibody (Rabbit anti-Mouse IgG HRP, #61-6520, Invitrogen; RRID:AB_2533933 1:5000 in TBS-T)) for 1 h at room temperature, before ECL detection (Milipore) and imaging using iBright 1500 (ThermoFisher Scientific). Band density was quantified using Image J software [92 (link)]. The original immunoblot image is presented in Additional File 1: Figure S8H.