Anti ambra1

The cells were washed twice with PBS containing 1 mmol/l phenylmethylsulfonylfluoride (PMSF). Next, the cells were scraped off the dishes and pelleted at 500×g for 10 min. The cells were subsequently lysed in cold lysis buffer (20 mmol/l Tris-HCl, 1 mmol/l EDTA, 150 mmol/l NaCl, 1 mmol/l EGTA, 1% Triton X-100, 2.5 mmol/l sodium pyrophosphate, 1 mmol/l β-glycerophosphate, 1 mmol/l Na3VO4, 1 µg/ml leupeptin and 1 mmol/l PMSF) and sonicated for 5 s. The lysates were clarified by centrifugation at 12000×g for 30 min at 4°C. Equal amounts of cell lysate were resolved by 8 or 15% SDS-PAGE. The membranes were blocked for 1 h in 5% powdered milk in TBST (10 mmol/l Tris-HCl, 150 mmol/l NaCl and 1% Tween-20). The membranes were subsequently immunoblotted using anti-Ambra1, anti-Beclin1, anti-LC3, anti-PARP, anti-Cathepsin D, anti-ATG5, anti-VPS34 and anti-β-actin antibodies (Cell Signaling Technology, Danvers, MA, USA). The PVDF membranes were incubated with a horseradish peroxide-conjugated anti-rabbit IgG antibody (Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. Immunoreactive bands were exposed to film (Kodak, Rochester, NY, USA). The band intensity was semi-quantified using BandScan software after scanning the blots (V300; EPSON, Tokyo, Japan).