Quantification of Apoptosis Markers

Cal-27 cells treated for 24h or 48h with vehicle, CIDD-24, CIDD-99, and CIDD-111 at the indicated concentrations were harvested and lysed in Laemmli Lysis Buffer. Cell lysates (40μg) were separated using electrophoresis in 10% SDS-PAGE, separated proteins were transferred to PVDF membrane, and membrane blocked in 5% milk solution. Rabbit polyclonal antibodies (Cell Signaling, Danvers, MA, USA) against cPARP (#5625S; 1:1250), Bip (#3177S; 1:1000), and CHOP (#2895T; 1:1000) and rabbit monoclonal anti-α/β-tubulin antibody (Cell Signaling, #CS2148; 1:1,000) were diluted in 6ml diluent (1% milk in PBS-0.1% Tw-20) and incubated overnight at 4°C (12 (link)). Membranes were washed with PBS-Tw-20, incubated with ECL Plus detection solution (GE Healthcare, South San Francisco, CA, USA) for 1 min, and signal was detected by radiographic exposure for 30 seconds.