Anti lc3 polyclonal antibody

Anti-LC3 polyclonal antibody (Novus Biologicals, Centennial, CO, USA, NB100-2220) was used for detecting autophagy. Cell lysates were separated on NuPAGE 12% Bis–Tris polyacrylamide gel (Invitrogen, Carlsbad, CA, USA) and transferred to Immobilon-P Transfer Membrane (Millipore, Bedford, MA, USA; pore size 0.45 µm). Membranes were blocked in Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and 5% nonfat dry milk (BioRad, Hercules, CA, USA) for 1 h, probed overnight at 4 °C with primary rat Anti-LC3 polyclonal antibody in TBS-T containing 1% nonfat dry milk and incubated for 1 h with rabbit anti-rat peroxidase-conjugated secondary antibody (DAKO, Glostrup, Denmark) at room temperature. Peroxidase activity was detected with SuperSignal West Femto Maximum Sensitivity Chemiluminescent Substrate (Pierce, Rockford, IL, USA) using a Lumi-Imager (Roche Diagnostics, Mannheim, Germany).

Capsaicin (CAP), N-Acetyl cysteine (NAC) and 3-methyladenine (3-MA) were purchased to Sigma (St. Louis, USA). The inhibitors E64 and pepstatin were purchased to Roche Diagnostics (Mannheim, Germany). Primary anti-p62, anti-pAkt and antibodies were from Cell Signalling Technology (Danvers, MA, USA) and the anti-LC3 polyclonal antibody was obtained from Novus (England, UK). Peroxidase labeled secondary anti-mouse IgG was from Sigma (St. Louis, USA) and anti-rabbit IgG was from Calbiochem (San Diego, USA).