Bcl 2 bs 4563r

Western blotting was performed as described previously.²⁶ The brain tissues or cells were homogenized in RIPA lysis buffer (P0013; Beyotime) with protease/phosphatase inhibitor cocktail (P0044/P8340; Sigma Aldrich). Lysates were centrifuged at 14,000 g for 30 min at 4°C. Total protein concentrations were measured using the Bradford assay kit (P0006; Beyotime). Supernatants were mixed with an equal volume of 2× loading buffer (Santa Cruz) and boiled for 10 min. Protein samples were electrophoresed and separated in a 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gel and transferred onto a 0.45 μm nitrocellulose membranes at 100 V for 1 h. After blocking by 5% nonfat milk, the membrane was incubated over night at 4°C with the following primary antibodies: p‐Akt (2965; CST), t‐Akt (9272; CST), p‐Smad1 (9516; CST), p‐Smad2 (3104; CST), p‐Smad3 (9520; CST), Bcl‐2 (bs‐4563R; Bioss), Bax (bs‐4564R; Bioss), p‐Nrf2 (ab76026; Abcam), Nrf2 (ab76026; Abcam), tubulin (sc‐8035; Santa Cruz), or β‐actin (sc‐1265; Santa Cruz). Membrane was washed with Tris Buffered Saline with Tween 20 (TBST) three times and incubated with horseradish peroxidase‐labeled secondary antibody for 1 h at room temperature. The immunoblotting was detected using an enhanced chemiluminescence detection kit (WBKLS0500; Millipore). Grayscale analysis was performed using the ImageJ software.