<p>(A) A co-IP assay to detect β-catenin interaction with Gal4-AR LBD in 293 cells. Proteins were immunoprecipitated with an anti-Gal4 antibody in lysates from 293 cells treated with or without indicated concentrations of DHT and MJC13. AR LBD was precipitated and blots probed for AR or β-catenin +/- DHT and MJC13. Inputs are shown at bottom. (B) A mammalian two-hybrid assay assessing the DHT-dependent activity of a Gal4-mediated luciferase reporter in the presence of a Gal4-AR LBD fusion with and without Vp16-β-catenin and 30 μM MJC13. MJC13 significantly (***p < 0.001) inhibits hormone-dependent Vp16 β-catenin/Gal4-AR LBD interaction in 293. Both conditions in the presence of Vp16 β-catenin were significantly (p < 0.001) enhanced as compared to hormone-dependent activity in the presence of Gal$-AR LBD alone. (C) 52KO MEFs were co-transfected with FKBP52, β-catenin (S33A), wild type AR, the AR-inducible luciferase reporter plasmid, and the constitutively active β-galactosidase reporter plasmid. After a 1 hour soak with a range of concentrations of MJC13, cells were induced with 10 pM DHT or ethanol for 16 hours. Following cell lysis, AR expression was assessed by luciferase assay. The data represent the average reporter expression (luciferase activity/β-galactosidase activity +/- standard deviation) of four replicates. MJC13 significantly (p < 0.001) inhibited hormone-dependent activity at all concentrations at or above 1 μM. MJC13 significantly inhibited hormone-independent activity at the 2.5 μM (p < 0.05), 5 μM (p < 0.01), and 10 μM (p < 0.05) concentrations.</p
