Fig. 4.

Skp2 mediates AR degradation via ubiquitination. A, B: Skp2 positively regulates AR ubiquitination. 293T cells were transfected with Myc-Skp2 (or shSkp2), Flag-AR and His-Ub plasmids for 48 hr, MG132 at 20 μM was added 3 hr before harvest. Ni-NTA beads were incubated with cell lysates, and the elution was subjected to immunoblotting analysis. C: Skp2 knockdown decreases endogenous ubiquitinated ARinC4-2B cells. C4-2B cells with Skp2 knockdown or scrambled sequence were used for immunoprecipitation with anti-AR or anti-ubiquitin antibody, normal rabbit IgG or mouse IgG were used as a control. Immunoprecipitated proteins were subjected to immunoblotting with anti-ubiquitin or anti-AR antibody. D: Skp2 knockdown restores AR expression and decreases A Rubiquitination in PC3 cells. AR and His-Ub were transfected into PC3 cells with Skp2 knockdown or control. The ubiquitination assay was performed as (A). E: AR mutation K847R abrogates Skp2-mediated AR ubiquitination. AR wild type, AR K845R or K847R mutants were transfected into 293T cells along with His-Ub, Myc-Skp2, and ubiquitination assay was performed as described previously.