Nuclear fractions were collected from 293-EcR and EcR-GR using NE-PER nuclear extraction reagents. Gel shift assays were run using the Lightshift chemiluminescent electrophoretic mobility shift assay kit. Double-stranded 5′-biotinylated oligonucleotides (5′-GGTCACTGCAAGAATGAACATTGAACTTTGGACTATAC-3′) corresponding to the wild-type sequence of GRE #1 was used as a probe. Following end-labeling with biotinylated UTP, complementary oligonulceotides in equimolar amounts were heated to 95°C for 1 min, cooled to 65°C, and then stored at −20°C. Binding reactions were performed at a 20 μl volume containing 20 fmol labeled probe, 5 μg nuclear proteins, 10 mM Tris, pH 7.5, 50 mM KCl, 1 mM dithiothreitol, 5 mM MgCl2, 2.5% glycerol, 0.05% NP-40, 1 μg herring sperm DNA, and 1 μg bovine serum albumin. Where indicated, 4 pmol of unlabeled competitor probe was added to reactions. For supershift experiments, 1 μg anti-glucocorticoid receptor antibody (BuGR2; Calbiochem) was added 10 minutes after addition of biotinylated probe and nuclear extract and incubated for an additional 20 minutes at room temperature. Reactions were then loaded onto an 8% TBE gel in 22.25% Tris, pH 8.4, 22.25% boric acid, 0.5 mM EDTA
