Figure 4.

AR is targeted for Mdm2-mediated ubiquitylation and destruction. (A) To confirm Mdm2 catalyses AR ubiquitylation, COS-7 cells transfected with AR, His-tagged ubiquitin, and either wild-type Mdm2 or an Mdm2 ubiquitylase-dead mutant Mdm2_C462A was treated with 1 μM MG132 for 8 h prior to immunoprecipitation with nickel–agarose (His-ubiquitin-bound proteins) followed by immunoblotting with an anti-AR antibody. (B) To assess a role for the enzymatic activity of Mdm2 in AR destabilization, COS-7 cells were transfected as above and AR protein levels analysed by western analysis. (C) To examine potential cross-talk between p53 and AR pathways, the effect of c-abl kinase on Mdm2-mediated AR destruction was examined. COS-7 cells were transiently transfected with AR and combinations of Mdm2 and c-abl followed by western analysis using an anti-AR antibody.