FIG. 6.

Enhanced N-CoR binding of the AR acetylation site mutant in cultured cells. (A) Smad3 binds components of the HDAC regulatory complex (HDAC1, N-CoR). Smad3 or a Smad3 mutant with the carboxyl terminus deleted (Smad3ΔC) was transfected with the MMTV-LUC reporter into DU145 cells with either ARwt or the AR acetylation site mutant, and the cells were treated with DHT for 24 h. (B) The binding of N-CoR to the AR was determined by IP-Western blotting. HEK293 cells transfected with expression vectors for FlagN-CoR and either ARwt or the AR acetylation site mutant were assessed by Western blotting in the presence of DHT for expression levels. Similar levels of AR are shown by Western blotting. Equal amounts of cell extracts were subjected to IP with anti-Flag antibody (M2) to precipitate N-CoR and Western blotting performed for Flag (N-CoR) and the AR. AR immunoreactivity is detected in the N-CoR IP. +, present; −, absent; GDI, guanine nucleotide dissociation inhibitor.