Fig. 2.

BMI1 stabilizes AR via competitive inhibition of MDM2-mediated ubiquitination. a C4-2 cells were transfected with siBMI1 or scramble and at 24 h post transfection, cells were treated with 10 µg ml⁻¹ CHX for the time indicated. Total cell lysates were blotted for BMI1 and AR, while GAPDH served as loading control. Zero-hour time points of both treatments were immediately post transfected. *P < 0.05 vs. Scramble + CHX, mean ± SEM. b C4-2 cells transfected with siBMI1 were treated as labeled at 24 h post transfection for 48 h, vehicle was used as the control; 20 µM MG132, 10 mM NH₄Cl, and 200 µM chloroquine were used. Total cell lysates were blotted for AR and BMI1, while GAPDH served as loading control. c C4-2 cells were transfected with siBMI1, siMDM2, or both as indicated; at 12 h post transfection, cells were treated with MG132 (20 µM) for another 36 h, and cells were then lysed and subjected to immunoprecipitation using anti-AR antibody, followed by immunoblotting with indicated antibodies. d BMI1 or BMI1 + MDM2 were knocked down using siRNA as indicated. Total cell lysates were immunoblotted for BMI1, AR, MDM2, and GAPDH. e C4-2 cells were transfected with siBMI1 or scramble. Twelve hours after transfection, MDM2 inhibitors were used to treat cells as indicated for another 36 h, followed by immunoblot analysis with indicated antibodies. GAPDH was used as a loading control. f Purified AR-NTD and GST-MDM2 proteins were incubated with purified His-BMI1 protein at indicated concentrations (0, 2, or 10 µg) at 4 °C for 12 h, followed by GST pull-down assay. The blots were probed with indicated antibodies. All experiments were biologically repeated at least three times. Representative images are shown