Anti atg4a

Total proteins were extracted using RIPA lysis buffer on ice, electrophoresed on 12% SDS-PAGE gels (Bio-Rad) and blotted onto nitrocellulose membranes (Amersham Biosciences Corp, Sunnyvale, CA). The membranes were blocked with 5% non-fat milk powder at room temperature for 2 hours and incubated overnight with primary antibodies: anti-ATG4A (1:400) (Abcam, Cambridge, UK), anti-E-cadherin (1:2000) (BD Transduction Laboratories, Franklin Lakes, NJ), anti-N-cadherin (1:1000) (BD Transduction Laboratories), anti-vimentin (1:1000) (BD Transduction Laboratories), anti-Sox2 (1:500) (Abcam), anti-Oct4 (1:400) (Abcam), anti-Bmi-1 (1:400) (Abcam), anti-LC3I/II (1:1000) (Cell Signaling Technology, Danvers, MA) and anti-β-actin antibody (1: 2000) (Sigma-Aldrich). After three 5-min washes, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1: 2000) (Santa Cruz Biotechnology, Dallas, TX) for 2 hours at room temperature and then washed again in TBS-T and visualized with an enhanced chemiluminescence kit (ECL-kit) (Santa Cruz Biotechnology). All of the experiments were performed in triplicate.

Human small-cell lung carcinoma H446 and LTEP-sm cells were purchased from the Tumor Cell Bank of the Chinese Academy of Medical Science (Shanghai, China) and cultured in RPMI 1640 medium containing 10% fetal bovine serum and ampicillin and streptomycin at 37ºC in a humidified atmosphere of 95% air and 5% CO₂. VP16–DDP-resistant H446 cells (H446/EP) were established and preserved in a final concentration of 1.5μg/ml VP16 and concentration of 1.25μg/ml DDP. Antibodies against LC3, P62, caspase-3, activated (cleaved) caspase-3 (c-caspase-3), PARP, cleaved PARP (c-PARP), Atg5 and GAPDH came from Cell Signaling Technology, (MA, USA). Anti-ATG4A, Anti-ATG4B, Anti-ATG4C and Anti-ATG4D antibodies came from Abcam. Bafilomycin A1 and 3-methyladenine (3-MA) came from Sigma Aldrich (St. Louis, MO).