Antibodies against cox 2

Western blot analysis was performed as described previously (Son et al., 2010a (link)). Monoclonal antibodies specific for β-actin were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies specific for NF-κB and Ref-1 were purchased from Santa Cruz, CA). Antibodies against COX-2, TNF-α, Bcl-2, PARP, C-Caspase 3, GAPDH, p62, and Nrf2 were purchased from Cell signaling Technology (Danvers, MA). Secondary antibodies and enhanced chemiluminescence substrate were purchased from Pierce Biotechnology (Waltham, MA). The blots were exposed to Hyperfilm (Amersham Bioscience, Piscataway, NJ). To generate cytoplasmic and nuclear protein, cadmium-treated cells were washed with cold PBS and scrape the cells in 1 ml PBS. Then the cells were suspended in 0.4ml lysis buffer (10 mM Hepes, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA) and lysed on ice for 15 min. Next 12.5 µl of 10% NF-40 was added, vortexed for 10 s, and spun for 1 min at max speed at 4°C. The supernatant is cytoplasm protein. 40 µl extraction buffer (20 mM Hepes (PH 7.9), 0.4M KCl, 1 mM EDTA and 1 mM EGTA) was added and then spun for 5 min at max speed at 4°C. The supernatant is nuclear protein. For lysis buffer and extraction buffer, protease inhibitor cocktail was added before use.