Electrophoretic mobility shift assay (EMSA) and determination of Kd values
PC3 cells were transfected with ARWT or AR∆DBD expression plasmid. Cell extract (5 µg) collected 36 h after transfection was incubated with γ32P ATP-labeled oligonucleotide probes with either ARWT or AR∆DBD consensus binding sites (Santa Cruz Biotechnology) in a buffer containing 20 mM HEPES, pH 7.9, 50 mM KCl, 0.1 Mm EDTA, 2 mM MgCl2, 2 mM spermidine, 0.5 mM dithiothreitol, 1 µg dI-dC and 10 % glycerol for 20 minutes at room temperature, followed by resolution of complexes on 7% native PAGE. For super-shift analysis, antibody against AR from Cell Signaling Technologies was used (Hellman & Fried, 2007).
The dissociation constants of the protein-DNA complexes were measured under equilibrium binding conditions. The volumes of the bands corresponding to free and bound DNA were quantified using ImageQuant software (version 5.2). The bound-DNA fraction (θapp) was calculated as the volume of the band corresponding to the bound DNA, divided by the sum of the volumes of the bands corresponding to free and bound DNA. Data were fit to a modified two-state binding equation to determine apparent dissociation constants for each protein-DNA complex as previously reported (Bird, Lajmi & Shin, 2002).