Fig. 3

BMI1 regulates AR signaling independently of PRC1. a RING1B or BMI1 was depleted by siRNA in C4-2 cells. After 48 h, cells were lysed and blotted for BMI1, RING1B, AR, PSA, and GAPDH. b C4-2 cells were treated with PRT4165 (100 μM) for 24 or 48 h. ubH2A, RING1B, RING1A, AR, PSA, and BMI1 were tested by western blot. Total H2A and GAPDH were used as loading controls. c C4-2 cells were transfected with siRNA duplexes as indicated. At 24 h post transfection, cells were infected by mouse flag-BMI1 lentivirus or vector lentivirus and incubated for an additional 48 h. Cells were then lysed and immunoblot analysis was performed with indicated antibodies. This anti-BMI1 antibody only recognizes human BMI1, but not mouse BMI1. GAPDH was used as a loading control. d GST-BMI1 or GST-BMI1ΔRING was incubated with RING1B for 12 h followed by IPs with an anti-RING1B antibody. e His-AR-NTD was incubated at 4 °C with GST-MDM2, GST-BMI1, GST-BMI1ΔRING, or GST-RING1B for 12 h, followed by GST pull-down and immunoblot analysis with anti-GST and anti-AR antibodies. f Purified AR-NTD and MDM2 proteins were incubated with purified BMI1∆RING protein at indicated concentration (0 or 2 µg) at 4 °C for 12 h, followed by MDM2 pull-down assay. The blots were probed with anti-AR, MDM2, or anti-BMI1 antibodies. g C4-2 cells were infected with shBMI1 lentivirus; at 24 h post infection, cells were infected with flag-tagged mouse full-length BMI1 lentivirus, mouse BMI1-RING lentivirus, or mouse BMI1ΔRING lentivirus as indicated, and cells were lysed after another 24 h and probed by indicated antibodies. All experiments were biologically repeated at least three times. Representative images are shown