Figure 4. CREB3L4 enhances AR transactivation through interacting with AR.

(a) Plasmids expressing hCREB3L4N (nuclear protein) and/or AR, under the pGL3b or human PSA gene promoters, were transfected into HEK293 cells, followed by treatment with or without 10-nM R1881. After 24 hr, cell lysates were subjected to luciferase assay. Luciferase activities were normalized to Renilla luciferase activities to adjust for transfection efficiency. Normalized activities are shown as means ± S.E. (error bars), n = 3, and expressed as folds-increase, relative to basal activity. (b) HEK293 cells were transfected with AR, and CREB3L4 expression vectors, and proteins immunoprecipitated (IP’ed) using an anti-AR and anti-Flag for CREB3L4 antibodies. IP protein was then resolved by SDS/PAGE and immunoblotted for the respective proteins indicated. (c) Interaction of endogenous AR and CREB3L4 in LNCaP cells. Cells were treated with 10-nM R1881 for 24 hr. Endogenous AR and CREB3L4 protein from LNCaP cells were precipitated with anti-AR or anti-CREB3L4 antibodies, and the interaction between these proteins determined by co-IP. (d) ChIP assay performed in LNCaP cells transfected with siCREB3L4, with R1881 treatment. Normal IgG was used as a negative control for IP. AR response element (ARE) of PSA gene promoter was amplified to determine ChIP’ed DNA, which was normalized to total input DNA.