Figure 5. PP-1 and Akt regulate pSer(213) of AR-v7 and Mdm2-mediated AR-v7 ubiquitination and protein degradation.

293T cells were transfected with wild type or mutant AR-v7 expression vector. A. Cells were also transfected with control, PP-1γ, PP-1γ mutant (H125A) vector; B. cells were treated with either vehicle or 5μM of TMC; or C. cells were transfected with control, cAkt or dAkt expression vector for 24 hours. AR-v7, PP-1γ, Akt and β-actin were detected by immunoblotting. 293T cells were transfected with wild type or mutant AR-v7 expression vector. D. Immunoprecipitation assays were performed using either control IgG or Mdm2 antibody. Associated proteins were detected by AR or Mdm2 antibody. E. Cells were also treated with 2μg/ml of MG132 for another 16 hours. In vivo ubiquitination assays were performed as described in the Material and Method section. All experiments were repeated at least three times with one set of results shown in the figure. F. LNCaP cells were transfected with expression vector encoding flag-tagged wild type or mutant AR-v7 and then treated with 5μM of TMC for 0 or 4 hours. G. 22Rv1 cells were transfected with expression vectors encoding flag-tagged wild type and mutant AR-v7 plasmid. Cells were also transfected with either cAkt or dAkt plasmid for 24 hours. Cells were fixed, immunostained with Flag-tag antibody and examined by fluorescence microscope.