Before treatments, the cells were synchronized by serum-free media. The treated cells were harvested and lysed with 20 mM Tris, 135 mM NaCl, 10% glycerol, 1% NP40, pH 6.8 for 30 min. Protein content of the cell lysates was measured with Pierce BCA Protein Assay (Thermo Scientific, 23,225)39 (link),40 (link). The samples were separated on 10% or 15% sodium dodecyl sulfate polyacrylamide gel and the SDS-PAGE was done by using Hoefer miniVE (Hoefer Inc.) Proteins were transferred to 0.45 µM PVDF membrane (Thermo Scientific, 88,518). The membranes were blocked with TBS Tween (0.1%), or 5% non-fat dry milk or 1% bovine serum albumin (Sigma-Aldrich, A9647)39 (link),40 (link). The membranes were incubated with the following antibodies: antiLC3B (SantaCruz, sc-271625), antip62 (Cell Signaling, 5114S), antiAMPK-P (Cell Signaling, 2531S), antiAMPK (Cell Signaling, 2603S), antip70S6K-P (Cell Signaling, 9234S), antip70S6K (SantaCruz, sc-8418), antiULK1-555-P (Cell Signaling, 5869S), antiULK1-777-P (Merck, ABC123), antiULK1 (Cell Signaling, 8054S) and antiGAPDH (Santa Cruz, 6C5), HRP conjugated secondary antibodies (Cell Signaling, 7074S, 7076S). The bands were visualized using a chemiluminescence detection kit (Thermo Fisher Scientific Inc., 32,106)39 (link),40 (link).
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