(a) DNA mobility gel shift assay. WT (W) or mutant (M) receptors were expressed in COS-7 cells and exposed to 10 nM (lanes 1 and 2) or 100 nM (lanes 3–12) DHT. Equivalent quantities of immunoreactive AR from the cell extracts were added to binding reactions containing ³²P-labeled synthetic ARE (lanes 1–10) or ERE (lanes 11 and 12) oligonucleotide sequences. Excess unlabeled ARE (lanes 5 and 6), ERE (lanes 7 and 8), or Oct (lanes 9 and 10) oligonucleotides were added as competitor DNA to demonstrate the specificity of the binding reaction. The dark band at the bottom represents unbound ³²P-labeled DNA. (b) Immunoblot of WT or mutant receptors used in gel shift assay. Five microliters of representative cell extract (used in the gel shift assay depicted in Figure 5a) was exposed to either 10 or 100 nM of DHT and was separated on an SDS-PAGE gel. AR protein was identified with a specific antibody (PG-21). (c) Quantification of binding to AREs. Receptors were expressed in COS cells, exposed to [³H]MB and equivalent quantities (50,000 dpm) of [³H]MB-AR complexes incubated with 150 pmol of either biotin-labeled natural ARE (an ARE from MMTV-LTR) or synthetic ARE in 2 independent series of experiments. Streptavidin-biotin–bound AREs were collected by centrifugation, and [³H]MB-labeled receptor bound to the AREs was quantified by scintillation counting. Known DNA binding–domain mutants (ΔF582, ΔR615) with severe impairment of DNA binding were used for comparison. In the right panel, background counts were lowered by treating the lysate with dextran-coated charcoal and by centrifugation at 100,000 g for 1 hour. Assays using mock-transfected cells showed minimal background activity. Each data point was the mean of 2 experiments, and bars indicate their range.