Ab109865

Cell lysates were prepared in RIPA Lysis and Extraction Buffer (#89900, Thermo Fisher Scientific) with Halt™ Protease Inhibitor Cocktail (#78425, Thermo Scientific). Equal amounts of protein (100 ug) were loaded in a well of SDS-PAGE gel, transferred to a nitrocellulose membrane subsequent to electrophoresis and probed with mitochondrial complex proteins followed by SuperSignal™ West Pico PLUS Chemiluminescent Substrate (#34577 Thermo Scientific). Antibodies used were Complex I(Abcam # ab109721), Complex II (abcam #ab109865), Complex III (ab109862) and complex V (ab109715). All antibodies were raised against human complexes and were used 1:1000 dilution.

Lysates were either made from mitochondrial fractions and whole cells. Fractions were collected in sucrose buffer (50 mM sucrose, 10 mMKCl, 20 mM HEPES, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA [Sigma, E8145], protease inhibitor [Roche, 4693132001], pH 7.4) on ice. To break the plasma membrane, the cell suspension was passed through a 26-gauge needle (Sigma, Z192392) ~30 times. Separation of the mitochondrial and cytoplasmic fractions was performed using differential centrifugation, as shown previously [60 (link)]. Primary antibodies: ACTB (Abcam, ab8826) 1:5000, ATP5B (Abcam, ab14730) 1:5000, ERK1/2 (Cell signalling, 9102S) 1:1000, pERK1/2 (Y202/Y204) (Cell signalling, 9101) 1:500, SDHA (Abcam, ab109865), TSPO (Abcam, ab109497) 1:4000, USP30 (Enzo, MBL-PW0975) 1:1000, VDAC1 (Abcam, ab14734) 1:2000, 1:10000 Vinculin (Abcam, ab129002) and 1:1000 TFEB (Cambridge Bioscience, A303-673A). Imaging was carried using an ECL based method (GE, RPN2133) on a a Bio-Rad ChemiDoc MP Imaging System, following incubation for 1 h with either anti-rabbit conjugated HRP 1:4000 (Dako, P0447), or rabbit anti-mouse conjugated HRP (Dako, P0448), or goat anti-rat IgG conjugated HRP 1:4000 (Abcam, ab97057). Densitometric analysis was performed using the ImageJ software. Values were normalised to ACTB loading control for whole-cell lysates and ATP5B for mitochondrial fractions.

Cell or tissue lysates of GP and SOL from cSix1KO and control mice (20–40 μg) were denatured in Laemmli buffer, separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. Membranes were blocked in 50 mM Tris-HCl pH 7.6, 137 mM NaCl and 0.1 % (v/v) Tween-20 containing 10 % (w/v) skimmed milk or 5 % (w/v) BSA for 1 h at room temperature and incubated overnight at 4 °C with the indicated primary antibodies (Complex I, NADH dehydrogenase, ab14713, Abcam (Cambridge, UK); Complex II, succinate dehydrogenase, ab109865, Abcam (Cambridge, UK); cytochrome bc1 complex, ab110252, Abcam (Cambridge, UK); Complex IV, cytochrome C oxidase, ab14744, Abcam (Cambridge, UK); Complex V, ATP synthase, ab14748, Abcam (Cambridge, UK); hexokinase II, Sc-6521 (Santa Cruz Biotechnology); glycogen synthase 1, CST #3893, Cell Signaling Technology; AS160, 07-741, Millipore; GLUT4, kind donation from Geoffrey Holman, University of Bath); lamin B, sc-6216 (Santa Cruz Biotechnology); β-tubulin, 05-661 (Millipore); Six1, HPA001893 (Sigma). Detection was performed using horseradish peroxidase conjugated secondary antibodies and enhanced chemiluminescence reagent.