Fig. 3.

FBXL2 plays a role in ALZ003-induced AR ubiquitination. A. Effect of ALZ003 on protein ubiquitination and ER stress. After treatment for indicated time points, cell lysates were prepared and subjected to western blotting using the indicated antibody. Lower panel is the quantitated results. Experiments were performed three times independently, and data were expressed as mean ± s.e.m. P-value was determined by Student's t-test with the Control group. (*p < 0.05, **p < 0.01, ***p < 0.001). B. After treatment for 24 h, cell lysates of U87MG were immunoprecipitated using the anti-AR antibody, and the immune complex was analyzed by western blotting using the anti-ubiquitin antibody. Lower panel is the quantitated results, and the ubiquitin signal ranged from 100 kDa to the top of membrane was quantified. Experiments were performed three times independently, and data were expressed as mean ± s.e.m. (*p < 0.05). C. The association of AR with the ubiquitin E3 ligase. AR-precipitated immune complex was analyzed by western blotting. Experiments were performed three times independently, and data were expressed as mean ± s.e.m. (***p < 0.001). D. Effect of FBXL2 on AR expression. After transfection with Flag-FBXL2 for 24 h or FBXL2 knockdown (E) for 3 days (Left), AR expression was estimated. Experiments were performed three times independently, and data were expressed as mean ± s.e.m. (**p < 0.01, ***p < 0.001). E. After FBXL2 knockdown for 48 h, U87MG cells were treated with ALZ003 for 24 h. AR-precipitated immune complex was analyzed by western blotting using the anti-ubiquitin antibody. Experiments were performed three times independently, and data were expressed as mean ± s.e.m. (***p < 0.001).