(A) TR4 interaction with AR in the mammalian two-hybrid system. PG5-Luc (3.5 μg), the luciferase reporter gene containing five copies of GAL-DBD binding sites, was cotransfected with two fusion proteins, GAL4-TR4E, and VP16-AR. After 16–18 hr transfection, 1 nM DHT was added and ethanol was used in control groups. After 24 hr treatment, cells were harvested for dual luciferase assay. (B) TR4 interaction with AR in modified mammalian one-hybrid system. A total of 3.5 μg of PG5-Luc and 3 μg of GAL4-TR4E was cotransfected in the presence of 1 μg of pSG5AR (lanes 2–9), pSG5GR (lane 10), pSG5PR (lane 11), or pSG5ER (lane 12). Cells were treated as indicated. Transfection was performed by a modified calcium phosphate precipitation method. The pRL-TK plasmid was cotransfected for normalization of transfection efficiency.