FIG. 2.

Interaction of hPRA-selective peptides with hPRA and hPRB in vivo. (A) Schematic of the mammalian two-hybrid assay. (B) Sequences of peptides which were fused to the Gal4DBD and used in the mammalian two-hybrid assay. These peptides were isolated by affinity selection from an X₇LXXLLX₇ phage display library using R5020-activated hPRA. (C) HepG2 cells were transiently transfected with 1,000 ng of 5XGal4-TATA-LUC reporter, 200 ng of pBKC-βgal, either 400 ng of VP16-hPRA, VP16-hPRB, or VP16 alone, and either 400 ng of a vector encoding Gal4DBD alone or Gal4DBD fused to a peptide; 1,000 ng of pBSII-KS was used to bring the total amount of DNA per triplicate up to 3 μg. At 24 h after transfection, cells were treated with no hormone or with 10⁻⁷ M R5020 for 24 h. Transcriptional activity was assayed on the 5XGal4-TATA-LUC reporter and represents an indirect measure of the binding of the fusion proteins. Transfections were normalized for efficiency using an internal β-galactosidase control plasmid (pBKC-βgal). The data are represented as fold induction over the no-peptide response for each condition tested, which was set to 1.0. Each data point represents the average of triplicate determinations of the transcriptional activity under the given experimental conditions (n = 3).