Androgens and OTUB1 regulate RhoA activity and p53 protein levels in PCa cells. (A) Phospho-protein array analysis of changes in protein phosphorylation in LNCaP-FGC cells transfected with control siRNA or an OTUB1 targeting siRNA, treated or not with DHT. Left panel shows the effects of DHT on siRNA control transfected cells and in the right panel the effects of different siRNAs on DHT treated cells are compared. Measurements were performed in duplicates. Students´ t test was applied to evaluate the statistical significance of the phosphorylation changes consequence of DHT (left panel) and OTUB1 depletion (right panel). p < 0.05 was considered as significant (*). (B) Analysis of p53, MDM2, OTUB1 and Beta-actin expression by western blot in extracts obtained from LNCaP-FGC cells transfected with siRNA targeting OTUB1 or siRNA control (upper panel) and OTUB1-WT, OTUB1-C91S variant or empty vector (lower panel) treated with or without DHT. (C) RhoA activity assay on cell extracts purified from LNCaP-FGC cells stimulated with DHT at different time points as indicated. (D) RhoA activity assay as in (C) using extracts from LNCaP-FGC cells transfected and treated as indicated.