Six1 hpa001893

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.