Mitochondrial Complex I Activity Assay

Assays were performed as in ref. ^{71 (link)}. Mitochondria were purified from ten thoraces of 7–10-day-old male flies and BN-PAGE was performed using NativePAGE gels from Life Technologies, following the manufacturer’s instructions. Mitochondria were suspended in NativePAGE sample buffer (Life Technologies) supplemented with 1% digitonin and protease inhibitors, incubated on ice for 20 min and centrifuged at 20,000 × g for 30 min at 4 °C, the supernatant was recovered, G-250 (Life Technologies) sample additive and NativePAGE sample buffer was added before loading onto 3–12% precast Bis–Tris NativePAGE gels (Life Technologies). Electrophoreses was performed using the NativePAGE Running buffer (as anode buffer, from Life technologies) and the NativePAGE Running buffer containing 0.4% Coomassie G-250 (cathode buffer). Protein complexes were revealed by staining with Novex Colloidal Blue staining kit (Life Technologies). Complex I activity in native gels was performed by incubating gels in 0.1 mg/mL NADH, 2.5 mg/mL nitrotetrazolium blue chloride, 5 mM Tris-HCl (pH 7.4) overnight at room temperature. Gels were imaged using a BioRad station and densitometry was performed in ImageJ.