Rabbit monoclonal anti α β tubulin antibody

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.

Cells were treated for 48 h with vehicle control, 200μM, 400μM, 600μM, or 800μM thymol (final DMSO concentration of 0.1%) and then harvested and lysed in 1% Triton-PBS. Cell lysate concentration was determined by A280 reading using Nanodrop 3300 (Thermo, Scientific, Waltham, MA). Cell lysates containing equal concentration of protein (100units/A280) were separated using electrophoresis in 10% SDS-PAGE. SDS-PAGE separated proteins were transferred to PVDF membrane and membrane blocked in 5% milk solution. Anti-cPARP rabbit polyclonal antibody (Cell Signaling, Cat #5625S, Danvers, MA) was diluted 1:1250 and rabbit monoclonal anti-α/β-tubulin antibody (Cell Signaling, Cat #CS2148) was diluted 1:1000 in a total of 5ml diluent (1% milk in PBS-0.1% Tw-20) and incubated overnight at 4°C. The membrane was thrice washed with PBS-Tw-20 and incubated with ECL Plus detection solution (GE Healthcare, South San Francisco, CA) for 1min. Signal was detected by exposure to radiograph film for 30 seconds.