Cleavage parp

Protein samples were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, UK). Blots were blocked for 1 h in 5% milk/0.1% Tween 20 in phosphate buffered saline (PBS-T) and then incubated with primary antibodies (1: 1000) at 4°C overnight. Blots were then washed three times for 15 min in PBS-T, followed by incubation with secondary antibody (according to different primary antibodies, HRP-conjugated goat anti-mouse, anti-rabbit, and rabbit anti-goat IgG were used (1: 5000, Santa Cruz, Dallas, TX)) in 5% milk/PBS-T for 1 h, and then washed three times for 15 min in PBS-T. The membranes were briefly incubated with ECL detection reagent (Amersham Biosciences, Castle Hill, Australia) to visualize the proteins and were then exposed on X-ray film. Primary antibodies used were as follows: ATM (Cat# 600-401-398), and p-ATM (Cat# 600-401-400) were purchased from Rockland Immunochemical (Gilbertsville, PA, USA); cleavage-caspase-3 (Cat# 9661), cleavage-PARP (Cat# 5625P), γ-H2Ax (Cat# 9718), Bax (Cat# 2772s), and Bcl-2 (Cat# 2870), ATR (Cat# 2790), p-ATR (Cat# 2853S) were from Cell Signaling Technology (Beverly, MA); PARP (Cat# 7150), pro-caspase-3 (Cat# 7272), β-Actin (Cat# 1615), Chk1 (Cat# 377231), p-Chk1 (Cat# 2341), Chk2 (Cat# 8813), p-Chk2 (Cat# 2661) were from Santa Cruz (Dallas, Texas, US).

NCI-H23 and A549 cells were lysed with RIPA lysis solution (Beyotime) containing a mixture of protease inhibitors. The cell protein lysis product was separated by 10% SDS-PAGE electrophoresis, transferred onto a 0.22 mm PVDF membrane (Millipore), and detected with a specific antibody. A chemiluminescent substrate was added to the specific strip and quantified with densitometry (Quantity One software, BioRad). The GAPDH antibody was used as a control. Anti-Caspase-3 antibody, cleavage Caspase-3, poly (ADP-ribose) polymerase, cleavage PARP, BAX, BAK, cyclin D3 and CDK4 (1:1000) were purchased from Cell Signaling Technology. Anti-SIRT6 antibody was purchased from Abcam. BCL-2 and BCL-XL antibody were purchased from Proteintech.