Fig. 5.

Ser-81 phosphorylation is not required for AR transcriptional activity. RLU, relative light unit. (A and B) HeLa cells were transfected overnight with pRL–CMV (2.5 ng) and ARE₄–luciferase (50 ng) reporters, together with AR WT (50 ng) or AR S81A mutant (50 ng) expression vectors, and then incubated in 5% CSS medium with DHT (10 nM) for 24 hr, as indicated. The cells were harvested and divided for luciferase assay (A) or immunoblotting (B). (C) An experiment similar to that shown in A was performed with 293T cells. (D) LNCaP cells were split in RPMI medium 1640 plus 5% CSS for 2 days and then treated with 1 nM DHT for different time points, and equal amounts of proteins were immunoblotted as indicated. (E) LNCaP cells were split in RPMI medium 1640 with 5% CSS for 2 days and then treated with 1 nM DHT for 2–24 hr, and total RNA from duplicate plates was analyzed on Affymetrix U133A GeneChip array. Androgen-stimulated genes were clustered into early responsive (Left) and late responsive (Right) groups. (F and G) LNCaP cells were split in RPMI medium 1640 with 5% CSS for 2 days and then treated with DHT (1 nM) (■) or vehicle (♦) for 2–24 hr, without (F) or with (G) cycloheximide (CHX) (10 μg/ml). Total RNA was isolated for real-time RT-PCR analysis of PSA and PLZF gene expression versus 18S rRNA, and values were normalized to levels before DHT addition. (H) 293T cells were transfected with 100 ng of WT AR or single-site (Ser/Thr) mutant expression vectors, together with 100 ng of activated Cdk1 (Cdk1-AF) expression vector or empty pCDNA3.1 vector. After overnight transfection, the cells were incubated in 5% CSS medium with 10 nM DHT for 24 hr and harvested in 2% SDS, and equal amounts of total protein were immunoblotted for AR.