Figure 5.

Lyn knockdown induces AR degradation via the proteasome. (a) Lyn knockdown decreases AR expression at the protein but not mRNA level. LNCaP and C4-2 cells were treated with 10 nM of Lyn or control siRNA. Total proteins were extracted and western blots were performed using Lyn and AR antibodies, Vinculin antibody was used as a loading control (left panel). RNAs were extracted and qRT-PCR was performed to evaluate the expression level of AR, which was normalized by GAPDH (right panel). (b) Lyn expression stabilizes AR. LNCaP cells transfected with 10 nM Lyn or control siRNA (left panel) and stably transfected cells LNCaP^Lyn and LNCaP^Mock (right panel) were treated with 10 μM cycloheximide for indicated time period. Total proteins were extracted and western blots were performed using AR and Lyn antibodies, Vinculin was used as a loading control. Intensity of AR was quantified using ImageJ software and normalized to vinculin expression. (c) Lyn expression protects AR from proteasomal degradation. LNCaP cells were transfected with 10 nM Lyn or control siRNA and were treated with 10 uM MG-132 for 6 h. Total proteins were extracted and western blots were performed using Lyn and AR antibodies, vinculin was used as a loading control. (d) Lyn knockdown induces dissociation of AR from Hsp90 and increases AR ubiquitination. LNCaP cells were transfected with 10 nM Lyn or control siRNA, and immunoprecipitation of AR was performed followed by western blot using ubiquitin, Hsp90 and AR antibodies (left panel). Input was blotted with AR, Lyn and Hsp90 antibodies and Vinculin antibody was used as a loading control (right panel).