Ab96134

Cells were washed with phosphate-buffered saline. Radioimmunoprecipitation assay protein isolation buffer supplemented with a protease inhibitor was used to lyse the cells [48 (link)]. A bicinchoninic acid protein assay kit with bovine serum albumin (BSA) as the standard was used to determine the protein concentrations in the cell lysates [49 ]. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to separate the proteins, which were then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% dry milk (nonfat) or BSA in Tris-buffered saline and 0.05% Tween 20 (TBS-T) for 1 h, and then were incubated with specific primary antibodies in 5% milk or BSA at 4 °C overnight. The membranes were washed three times with TBS-T and then incubated with secondary immunoglobulin G for 1 h at room temperature. Chemiluminescence was detected using ECL Western blotting substrate (Thermo) and visualized using an Amersham Imager 600. The following primary antibodies from Abcam were used at 1:1000 dilutions: Atg5 (#ab108327), Beclin1 (#ab207612), p62 (#ab96134) and LC3II (#ab192890) [50 ].