Fig. 2. AR binds to importin- through its hinge region in vitro and in mammalian cell extracts. (A) AR constructs coding for the GST-tagged N-terminal transactivation domain (TAD), GST-tagged DNA-binding domain (DBD), GST-tagged hinge (H) domain, GST-tagged ligand-binding domain (LBD) or GST-tagged LBD and H domain together (H-LBD), were overexpressed and immobilised on glutathione-S-sepharose (GSH) resin. wtAR, wild-type AR. (B) After incubation with purified importin-, beads were washed, proteins eluted and separated, and probed for importin- by western analysis (1:2000). Equal loading of GST-AR domains was verified by Coomassie staining of an identical gel, with GST-AR domains marked by an asterisk. (C) AR was immuno-precipitated from COS-7 cells and probed for bound importin- by western blotting. Immunoprecipitation was carried out with and without androgen (10 nM R1881).