Figure 3.

K311 is important for AR protein stability and plays a critical role for its transcriptional activity. (A) LNCaP cells stably expressing either FLAG-WT AR or FLAG-AR K311R were subjected to treatment with 10 μg/ml cycloheximide (CHX) as indicated, followed by Western blotting with an anti-FLAG antibody. Band intensity was quantified and graph represents three independent experiments ± SEM. (B) Silencing of endogenous AR using siRNA targeting the 3΄ UTR (siUTR) sequence absent in exogenous AR carrying vectors causes reduction in AR protein in non-transduced LNCaP cells compared to scrambled control siRNA (siScr). Cells overexpressing lentiviral WT AR or AR K311R show increased levels of exogenous AR compared to non-transduced LNCaP cells after silencing their endogenous AR with siUTR treatment. (C) qRT-PCR analysis of AR target gene expression in LNCaP cells stably overexpressing WT or K311R AR after silencing endogenous AR. Cells were cultured in steroid depleted media for 72 h followed by 24h treatment with 10 nM DHT. Data is a mean of three independent experiments normalized to HPRT1 ± SEM. (D) AR was immunoprecipitated from the LNCaP cells stably overexpressing WT or K311R AR cultured in the presence or absence of 10 nM DHT followed by immunoblotting. (E) HEK293T cells were transfected with WT or AR K311R alongside a luciferase reporter plasmid under the control of an androgen response sequence (ARE3) of the PSA promoter with and without p300 overexpression. Cells were cultured in steroid depleted media for 48 hours before 24 h treatment ± 10 nM DHT. Data represent a mean of three independent experiments ± SEM. Luciferase activity was normalized to each AR alone.