Figure 2. PP-1 regulates AR-FL and AR-v7 protein degradation through proteasome pathway.

A. LNCaP95 (LN95), LNCaP and 22Rv1 cells were treated with 5μM of tautomycetin (TMC) for 0-24 hours. B. LNCaP95, 22Rv1 and 293T cells were transfected with expression vector for control, HA-tagged PP-1α, β or γ. C. LNCaP95, 22Rv1 and 293T cells were transfected with plasmid encoding control, HA-tagged PP-1γ or PP-1γ mutant (H125A). 293T cells were also transfected with AR-FL and AR-v7 expression vector (B and C). AR-FL, AR-v7, phosphor-AR(ser213), HA-tagged PP-1 and β-actin were detected by immunoblotting. D. LNCaP95 cells were treated with 50μg/ml of cycloheximide (CHX) plus either vehicle or 5μM of TMC for 0-24 hours. AR-FL and AR-v7 were detected by Western blotting. Densitometry analyses of AR-FL and AR-v7 levels were normalized to β-actin. Results were from triplicate experiments. E. LNCaP95, PC3(AR-FL), PC3(AR-v7) and LNCaP cells were treated with 0, 2 and 5μM of TMC plus vehicle, 100nM of epoxomicin (EPOX) or 2μg/ml of MG132 for 24 hours. AR-FL and AR-v7 proteins were detected by Western blotting. Experiments were repeated more than three times and only one set of results was shown.