Figure 3.

E2-Ligand binding of AR and ARA₇₀. (A) In vitro synthesized AR, ARA₇₀, and ER were quantitated by [³⁵S]methionine labeling. Equal molar concentrations of ER and AR were used for the [³H]E2 ligand binding assay. Three-fold molar ARA₇₀ was incubated with AR on ice for 1 hr before adding 50 nM [³H]E2. Two hundred-fold unlabeled E2 was used as a competitor to determine the specific binding. (B) E2-specific binding of full-length AR. AR was transcribed and translated in a rabbit reticulocyte lysate system. Aliquots of the lysate were then incubated with 50 nM [³H]E2 (87 Ci/mmol; 1 Ci = 37 GBq) in the presence or absence of 20-fold, 200-fold, and 500-fold unlabeled steroids. The final incubation volume was 100 μl. The values of duplicate assay tubes were within 10% of the average shown in the figure.