Rabbit anti cleaved caspase 8 antibody

CNE1 cells were seeded onto 90-mm petri dishes and treated with different concentrations of GSLL for 48 h. The cells were trypsinized, washed with PBS, and lysed with RIPA buffer on ice for 2 h. The cell lysates were centrifuged at 20,000×g and 4 °C for 30 min, and the supernatants were collected as the protein fractions. SDS-PAGE was performed, followed by semi-dry transfer onto a PVDF membrane using a Trans-Blot^® SD Semi-Dry Transfer Cell (Bio-Rad) at a constant voltage of 15 V for 40 min. The PVDF membrane was blocked with 5 % milk in Tris-buffered saline containing 0.1 % Tween-20 (TBST) for 1 h, incubated with a primary antibody (1:1000 in 5 % milk in TBST) at 4 °C overnight, washed with TBST, and incubated with the corresponding horseradish peroxidase (HRP)-associated secondary antibody (1:1000 in 5 % milk in TBST) at room temperature for 2 h. Primary antibodies used included: Rabbit anti-caspase 3 antibody (Code: #9662; Cell Signaling); Rabbit anti-cleaved caspase 8 antibody (Code: #9496; Cell Signaling); Rabbit anti-caspase 9 antibody (Code: #9502; Cell Signaling). Secondary antibody used included HRP-linked anti-rabbit antibody (Code: #7074; Cell Signaling). The presence of the target proteins was visualized with an electrochemiluminescence detection system using an Amersham ECL Western Blotting Detection Kit (Code: RPN2018; GE Healthcare).