Figure 5A-D, AR transcript levels were measured by RT-qPCR on LNCaP cells (A and C) or VCaP cells (B and D) transfected with siTRIM33 or the TRIM33 expression construct and treated with EtOH or DHT. Protein lysates were harvested at 72 h after transfection and subjected to western blot analysis with the indicated antibodies. Data information: In (A-D, data are presented as mean ± SD (n = 3 independent experiments). * P < 0.05, **P < 0.01, ns=not significant (two-way ANOVA).E and F, LNCaP cells transfected with siTRIM33 or NC were treated with cycloheximide (CHX). Protein lysates were harvested at the indicated time points for western blot analysis. AR protein levels were quantified and normalized to β-actin and then plotted relative to 0 h. Data information: In F, data are presented as mean ± SD (n = 3 independent experiments). * P < 0.05, **P < 0.01, ns=not significant (two-way ANOVA).G, LNCaP cells transfected with siTRIM33 or NC were treated with DMSO or MG132 for 8 h. Protein lysates were extracted and subjected to western blot analysis with the indicated antibodies.HA-Ub was expressed in LNCaP cells co-transfected with siTRIM33 (H) The cells were treated with MG132 for 4 h before harvesting. Endogenous AR was immunoprecipitated from cell lysates, and interacting proteins were detected by immunoblotting with the indicated antibodies.I, HA-Ub was expressed in LNCaP cells co-transfected with the TRIM33 expression construct (I). The cells were treated with MG132 for 4 h before harvesting. Endogenous AR was immunoprecipitated from cell lysates, and interacting proteins were detected by immunoblotting with the indicated antibodies.LNCaP cells transiently expressing myc-tagged Skp2 with Flag-TRIM33 were harvested and subject to immunoblot analysis with the indicated antibodies.K, LNCaP cells transiently expressing myc-tagged SPOP with Flag-TRIM33 were harvested and subject to immunoblot analysis with the indicated antibodies.L, shTRIM33- or shCtrl-LNCaP cell lines transfected with NC or siSkp2 were harvested 72 h after transfection and subjected to immunoblot analysis with the indicated antibodies.M, HEK293T cells were co-transfected with flag-TRIM33 and various myc-tagged AR constructs. AR-FL and AR truncated mutants were immunoprecipitated with an anti-myc antibody and then subjected to western blot analysis with the indicated antibodies.N, HEK293T cells were co-transfected with myc-AR-N and various flag-tagged TRIM33 constructs. AR-N was immunoprecipitated with an anti-myc antibody and then subjected to western blot analysis with the indicated antibodies.O, LNCaP cells were transfected with various flag-tagged TRIM33 constructs. AR-associated proteins were immunoprecipitated with an anti-AR antibody and then subjected to western blot analysis with the indicated antibodies.P, LNCaP cells were co-transfected with myc-Skp2 and various flag-TRIM33 constructs. AR and other proteins were detected by western blot analysis with the indicated antibodies.Q, AR pull-down was performed on protein extracts from MG132-treated HEK293T cells overexpressing myc-AR, HA-Ub, myc-Skp2, and various flag-tagged TRIM33 constructs. AR ubiquitination and specific protein levels were detected by western blot analysis with the indicated antibodies.