Anti caspase 3

1 × 10⁶ AGS cells were treated with the 201E4 mAb (20 μg/ml) and collected at 0, 12, 24, and 36 h after mAb 201E4 incubation. Cell lysate proteins (50 μg per lane) were loaded on SDS‐PAGE, transferred to PVDF, and probed with specific Abs, such as anti‐caspase‐8, anti‐cleaved‐caspase‐8/9 and anti‐Bak (Immunoway); anti‐caspase‐3, anti‐cleaved‐caspase‐3, anti‐PARP, anti‐AKT, anti‐pAKT, anti‐p44/42, anti‐p‐p44/42, anti‐p70 S6, anti‐PI3K p85, anti‐p53, anti‐Bcl‐XL and anti‐GAPDH (Cell Signalling Technology, USA), anti‐β‐action (ABclonal). All antibodies were diluted in 1% BSA‐TBST.

After treatment, cells were washed with cold PBS and then lysed adequately by RIPA buffer (Beyotime). Sodium dodecyl sulfate- (SDS-) polyacrylamide gel electrophoresis (PAGE) loading buffer was added to protein lysates, which were boiled and then clarified by centrifugation at 12,000 ×g for 10 min at 4°C. Next, the lysates were separated by 12% or 15% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. Membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20 and incubated overnight at 4°C with the following specific primary rabbit polyclonal antibodies: anti-caspase-3, 1 : 500; anti-JNK, 1 : 250; anti-p-JNK (phosphor-Thr183/Y185), 1 : 500; and anti-GAPDH, 1 : 1000 (ImmunoWay Biotechnology, Plano, TX, USA). Membranes were then incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit antibody (1 : 5000; ImmunoWay Biotechnology). Immunoreactive proteins were visualized using enhanced chemiluminescence, and the optical density of each band was measured and quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The commercial antibodies that were used included anti-E2F1 (catalog # AP7593b; Abgent, San Diego, CA, USA), anti-LRPAP1 (catalog # AP8529b; Abgent, San Diego, CA, USA), anti-LRP1 (catalog # AJ1448a; Abgent, San Diego, CA, USA), anti-Caspase-3 (catalog # YT0656; ImmunoWay, TX, USA), anti-GFP (catalog # AE012; ABclonal, Cambridge, MA, USA), and anti-actin (Sigma-Aldrich, St. Louis, MO, USA).
Dilution of the primary antibodies: anti-E2F1 (1:500), anti-LRPAP1 (1:500), anti-LRP1 (1:10000), anti-Caspase-3 (1:1000), anti-GFP (1:1000), and anti-actin (1:5000).