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).