Fig. 2

Loss of USP14 expression/function accumulates the poly-K48-ubiquitination of AR and promotes AR degradation. a Total RNAs were collected from MDA-MB-453 cells stably expressing USP14 shRNA or control shRNA and subjected to RT²-PCR analysis. Three independent experiments were performed. Mean ± S.D. (n = 3). b Total RNAs were extracted from MDA-MB-453 cells treated with IU1 for 24 h and subjected to RT²-PCR analysis. Three independent experiments were performed. Mean ± S.D. (n = 3). c Protein lysates were collected from MDA-MB-453 cells treated with the indicated doses of IU1, auranofin (Aur), or b-AP15 for 48 h. Western blot assay was used to detect AR protein level. d Protein lysates were collected from MDA-MB-453 cells exposed to IU1 (100 μM), Aur (1 μM), and b-AP15 (1 μM) in the presence or absence of bortezomib/ Velcade (50 nM) for 24 h and western blot assay was used to detect AR protein level. e, f Protein lysates were collected from MDA-MB-453 cells. Co-immunoprecipitation assay was performed to detect AR, USP14, and UCHL5 interaction. g Protein lysates were collected from LNCaP and MDA-MB-453 cells treated with UCHL5 siRNA for 48 h. Western blot assay was used to detect AR and UCHL5 protein level. h Protein lysates were collected from MDA-MB-453 cells stably expressing USP14 shRNA or control shRNA. Co-immunoprecipitation assay was performed using AR antibody beads, and immunoblotted for ubiquitin (Ub), K48-Ub, USP14, and AR. Cells were exposed to MG132 (10 μM) for 6 h before harvest. i Protein lysates were collected from MDA-MB-453 cells treated with IU1 for 48 h. Co-immunoprecipitation assay was performed using AR antibody beads, and immunoblotted for ubiquitin (Ub), K48-Ub, USP14, and AR. Cells were exposed to MG132 (10 μM) for 6 h before harvest