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. 2015 Sep 10;6(32):33743–33754. doi: 10.18632/oncotarget.5608

Figure 3. PP-1 regulates AR-FL and AR-v7 protein stability through Mdm2 ubiquitin ligase.

Figure 3

LNCaP95 (LN95) and 22Rv1 cells were: A. treated with 0-5μM of TMC for 24 hours or B. transfected with control or pooled PP-1α/β/γ siRNA for 48 hours or C. transfected with plasmid encoding control, PP-1γ or PP-1γ mutant (H125A) for 24 hours. AR-FL, AR-v7, phosphor-Mdm2(ser166), total Mdm2, phosphor-Akt(ser473), PP-1 and Akt proteins (only PP-1α was shown) were detected by immunoblotting. D. LNCaP95 and 293T(AR-v7) cells were transfected with control or Mdm2 siRNA and then treated with either vehicle or 5μM of TMC for 48 hours. AR-FL, AR-v7, Mdm2 and β-actin were detected by immunoblotting. E. 293T cells were co-transfected with AR-FL and AR-v7 expression vector. Cells were also transfected with control or Mdm2 plasmid in addition to control, PP-1γ or PP-1γ mutant (H125A) plasmid as indicated for 24 hours. AR-FL, AR-v7, PP-1γ and β-actin were detected by immunoblotting. 293T cell were transfected with AR-v7 F. or AR-FL G. expression vector. Cells were also transfected with ubiquitin vector plus either control or Mdm2 plasmid, and then treated with 2μg/ml of MG132 for 16 hours. In vivo ubiquitination assays were performed as described in the Material and Method section. All experiments (A-G) were repeated at least three times with one set of results shown in the figure.

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