Anti actin clone c4

Infected MC3T3-E1 cells were lysed in ice-cold lysis buffer consisting of 20 mM HEPES (pH 8), 5 mM EDTA, 10 mM EGTA, 5 Mm NaF, 10% glycerol, 1 mM dithiothreitol, 400 mMKCl, 0.4% Triton X-100, 20 mM sodium glycerophosphate, and a protease inhibitor cocktail (Sigma-Aldrich). Lysates were incubated on ice for 10 min and cleared by centrifugation at 13,000 g for 10 min. Protein concentration was determined by the bicinchoninic acid method (Pierce, Rockford, IL, USA) using bovine serum albumin as standard, and equal amounts of proteins were loaded onto electrophoresis gels. After separation, proteins were transferred to a nitrocellulose membrane (GE Healthcare, Little Chalfont, UK) and blocked for 1 h with 5% milk protein-0.05% Tween 20. Then, membranes were incubated with rabbit anti-MAP LC3B (Cell signaling), goat anti-BECN-1 or anti-p62 (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. After washing, the membrane was incubated with peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) (1:1,000 dilution), for 1 h. Protein bands were visualized on Hyperfilm ECL (GE Healthcare) by chemiluminescence. Equal loading was checked by Ponceau staining and by incubation of the blots with an anti-actin (clone C4; Santa Cruz Biotechnology).