Sc 365626

Whole-cell and nuclear extracts were prepared as previously described [35 (link)]. Western blot analysis was performed as described previously [29 (link)]. Briefly, cell extracts (10–40 μg/lane) were separated by 8–10% SDS polyacrylamide gel electrophoresis and electroblotting, and proteins were detected using antibodies against p-Nrf2 (ab76026, Abcam), Nrf2 (ab62352, Abcam), Keap1 (sc-365626, Santa Cruz Biotechnology), HO-1 (ADI-SPA-895-F, Enzo Life Sciences, NY, USA), SOD-1 (sc-11407, Santa Cruz Biotechnology), SOD-2 (sc-30080, Santa Cruz Biotechnology), lamin B1 (ab16048, Abcam), and β-actin (sc-47778, Santa Cruz Biotechnology). After detection with horseradish peroxidase-conjugated secondary antibody (anti-mouse or -rabbit), proteins were visualized using an enhanced chemiluminescence detection system (Santa Cruz Biotechnology) and an EZ-Capture ST imaging system (Atto, Tokyo, Japan). β-actin was used as the loading control. The densitometry data represent the mean ± standard error (SE) from three immunoblots and are shown as the relative density of protein bands normalized to the indicated protein (input Nrf2, input Keap1, actin, or lamin B1).

Whole-cell extracts (6–40 μg/per lane) were loaded onto 8–10% SDS polyacrylamide gels and separated by electrophoresis under reducing conditions. The proteins were then transferred by electroblotting and verified by using reversible staining with Ponceau S. The membranes were blocked using 3% non-fat dry milk in TBS-T (Tris-buffered saline and 0.2% Tween 20). The proteins were detected with antibodies for Nrf2 (sc-722, Santa Cruz Biotechnology, Dallas, TX, USA), p-Nrf2 (ab76026, Abcam, Cambridge, UK), KEAP 1 (sc-365626, Santa Cruz Biotechnology), and HO-1 (ADI-SPA-895, Enzo Life Science Inc., Farmingdale, NY, USA). The antibodies were diluted in TBS-T containing 3% dry milk and incubated with the membrane overnight at 4 °C. After washing with TBS-T, primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit, anti-mouse, anti-goat) and visualized by using the enhanced chemiluminescence detection system (Santa Cruz Biotechnology, Dallas, TX, USA) and BioMax MR film (Kodak, Rochester, NY, USA). The protein levels were compared to that of the loading control actin, or histone H1.