Fig. 2

PIAS1 together with SUMO3 facilitates ubiquitin-proteasome mediated AR degradation. (a) DU145 cells in a 12-well plate were transiently transfected with empty vector, AR or AR along with PIAS1 and GFP-SUMO3. Cells were then fixed at different transfection periods (24 h, 48 h, 72 h and 96 h), and stained for AR (red). Representative images of transfected cells were shown. (b) DU145 cells were transfected with plasmids as described in A. The AR mRNA or beta-actin mRNA levels were analyzed by reverse-transcriptional PCR at indicated time course after transfection (24 h, 48 h, 72 h and 96 h). (c) DU145 cells were transfected with plasmids as described in A. Whole cell lysates at indicated time course after transfection were generated together and immunoprepapited with anti-AR antibody. The Immunoprecipitate was detected by anti-AR (IP, top panel), anti-ubiquitin (IB, second panel), and anti-SUMO3 immunoblotting (IB, third panel). Whole-cell lysates (Input) were immunoblotted with anti-AR (fourth panel) or anti- actin (bottom panel) antibodies. (d) DU145 cells were cotransfected with empty vectors, AR or AR together with GFP-SUMO3 and PIAS1. Cells were then treated with or without MG132 (5 μM) for 16 h before cells were collected at 72 h or 96 h as indicated after transfection. Whole-cell lysates were immunoblotted with anti-AR or anti-actin antibodies