Figure 1.

Time course of PBP interactions with RAR and its requirement for RAR- and ER-dependent transcription. (A) Western blot of nuclear and cytoplasmic extracts with anti-PBP antibody, indicating specificity for a 220-kDa band. (B) Time course of PBP interaction with RAR after RA treatment. HeLa cells treated with 1 μM RA were harvested for immunoprecipitation with anti-RAR antibody at the indicated times. Immunoprecipitates were resolved by SDS/PAGE, transferred to nitrocellulose membranes, and probed with anti-PBP antibody. (C) Microinjection of anti-PBP antibody abolishes RA and estrogen-dependent transcription of transiently transfected reporter genes. Rat-1 cells were microinjected with expression vectors for RAR or ER as indicated and the corresponding LacZ reporter genes. Cells were coinjected with control IgG or anti-PBP IgG, treated with agonist, and assayed for LacZ expression 24 h later. Effects of anti-PBP were rescued by overexpression of wild type PBP (cmv/PBP), but not by PBP containing mutations in the LXXLL interaction motifs (cmv/PBP_mut). (D) Microinjection of anti-SRC-1 and anti-PBP IgG abolishes RA-dependent transcription in Rat-1 cells containing a chromosomally integrated RAR β2 reporter gene.