Mouse monoclonal anti β actin antibody clone ac 15

Cells were washed with PBS, scrapped in ice-cold PBS and centrifuged at 500× g for 5 min. The cell pellets were re-suspended directly in the reducing sample buffer (60 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulphate (SDS), 100 mM dithiothreitol and 0.01% Bromophenol Blue) in the presence of a complete EDTA-free protease inhibitor cocktail (Roche Diagnostics, Meylan, France). Proteins were separated on an SDS-polyacrylamide gel and electrotransferred to polyvinylidene difluoride membranes (Immobilon, Millipore, Dutscher, Brumath, France). Blots were blocked for 1 h with Tris-buffered saline-0.05% Tween 20 (TBS-T) supplemented with 5% nonfat milk and incubated overnight at 4 °C with a primary antibody. Filters were then washed in TBS-T, incubated for 45 min at room temperature with appropriate secondary antibodies conjugated to horseradish peroxydase and washed again prior to detection of signal with ECL plus chemilumiscent detection kit (Thermo Scientific). Primary antibodies used in this study were mouse monoclonal anti-LC3 antibody (MBL, Clinisciences, Nanterre, France) and mouse monoclonal anti-β-actin antibody (clone AC-15, Sigma-Aldrich). Original blots can be found at Figure S5.