|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 1. Schematic representation, expression and transactivating capacity on androgen-regulated promoters of GFP-AR proteins investigated. (A) As well as GFP-tagged wild-type AR, ARs containing a mutation in the DBD disrupting DNA-binding (Brüggenwirth et al., 1998), or in helix 3 (W741C) or helix 12 (T877A) of the AR LBD, which result in altered ligand specificity (Veldscholte et al., 1990; Hara et al., 2003) were studied. TAD, Transactivating domain; DBD, DNA-binding domain; LBD, Ligand binding domain. (B) Hep3B cells containing stably integrated GFP-AR expression constructs and AR expressing prostate cancer cell lines were cultured for 1 day in the absence (lanes 1-6) or presence of 1 nM R1881 (lanes 7-12). Cell lysates were prepared and subjected to western blotting. Western blots of cell lysates from Hep3B cells (AR negative) containing GFP-AR (lanes 1 and 7), GFP-AR(A573D) (lanes 2 and 8), GFP-AR(W741C) (lanes 3 and 9) or GFP-AR(T877A) (lanes 4 and 10) and LNCaP (lanes 5 and 11) and PC346 (lanes 6 and 12) using an anti-AR or ß-actin antibody. ß-actin expression was used as a loading control. (C,D) Co-transfection assays of GFP-AR and the mutants depicted in A with androgen-regulated promoter constructs (ARE)2-TATA-luciferase (C) and mouse mammary tumour virus (MMTV) luciferase (D) in the presence of 10–9 M R1881, 10–6 M bicalutamide (Bic), 10–6 M OH-flutamide (OH-F) or no ligand as indicated. Luciferase activity of the GFP-AR proteins is plotted relative to activity of GFP-AR in presence of 10–9 M R1881. Mean±s.e.m. of at least three independent experiments are shown.
|
