Immun blot pdvf membrane

Western blotting was carried out as previously described [25 (link)]with some modifications. Briefly, cells were lysed in lysis buffer (50mM Tris.HCl, pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate, 0.001% bromophenol blue, 100mM DTT, 10X protease inhibitor cocktail (Roche) and phosphatase inhibitors (Sigma)). Samples were heated to 95°C for 10 minutes and stored at -80°C. Lysates were subjected to SDS/PAGE on 15% Tris-Tricine Criterion (Bio-Rad, Hercules, CA) minigels followed by transfer to 0.2-μm-pore polyvinylidene difluoride membrane (Immun-Blot PDVF membrane; Bio-Rad). Membranes were probed with antibodies to mouse anti-human LC3 (2G6, nanoTools Antikörpertechnik GmbH, Teningen Germany) and mouse anti-human β–actin (Sigma). Following exposure to HRP-conjugated goat anti-mouse secondary antibody (Cell Signaling Technology, Danvers, MA, USA), proteins were visualized by enhanced chemiluminescence using Supersignal West Pico chemiluminescent substrate (ThermoScientific, Rockford, IL, USA).

A western blot survey of FAO and respiratory chain protein expression in mouse heart, liver, muscle, and brain was performed by homogenizing approximately 100 mg of tissue in ice cold PBS using a Virtis HandiShear (The Virtis Company, Gardinier, New York) homogenizer followed by sonication using a microtip, and centrifugation at 30 minutes, 14,000 x g, at 4°C. The supernatant (100 μg protein) was loaded onto an SDS-PAGE gel and transferred to Immun-Blot PDVF membrane (BioRad, Hercules, California). MitoProfile Total OXPHOS Rodent WB antibody cocktail (Abcam, Cambridge, MA), citrate synthase (C-20) (Santa Cruz Biotech Inc, Dallas, TX), VLCAD, and MCAD were used for visualization of western blots of 4–15% SDS polyacrylamide gels.