FIG. 1.
Specific binding of the GR to the POU domain of Oct-1 in vitro is abrogated by C-terminal truncation into the DBD. (A) Schematic presentation of in vitro-translated rat GR fragments used in panels B and C. The positions of the DBD, ligand binding domain, and heat shock protein binding domains of GR are diagrammed. (B) In vitro-translated Dex-treated GR, CREB, and firefly luciferase (Luc.) were tested for binding to GST–Oct-1 POU by a pulldown assay. Levels of binding (lanes 1 to 3) were compared to those of 10% of the receptors added to the incubations (lanes 4 to 6). The migrations of molecular mass markers are indicated to the left of the autoradiographs. (C) In vitro-translated GR fragments were tested for binding to GST–Oct-1 POU. GRWT and X795, which contain intact ligand binding domains and which were complexed with heat shock proteins in the absence of ligand, were tested for Oct-1 POU binding in both the presence (+) and absence (−) of bound steroid (Dex). SDS-polyacrylamide gels were exposed equally to compare specific levels of binding (lanes 1 to 6) to those of 10% of the in vitro-translated proteins added to the incubation (lanes 7 to 12). (D) In vitro-translated full-length and C-terminally deleted GRs produced by transcription of a restricted DNA template were tested for binding to immobilized recombinant GST–Oct-1 POU. Bound untreated and Dex-treated full-length receptors are shown in lanes 1 and 2, respectively, while the C-terminal truncations are shown in lanes 3 to 5. Levels of binding of proteins are compared to levels of binding of 10% of the input proteins shown in lanes 6 to 10.