FIG. 9.
The binding of GR to Oct-1 POU in solution is sensitive to the presence of a GRE. (A) Following the incubation of GR with GST–Oct-1 POU, binding was challenged with a consensus GRE (lane 2), highly sheared CT DNA (lane 3), or nonspecific oligonucleotides encoding an IAP enhancer core (lane 4) or a binding site for the Ku autoantigen on the C3H MMTV LTR (lane 5). GST–Oct-1 POU binding was determined by exposure of an SDS-polyacrylamide gel and compared to that of 10% of the in vitro-translated GR added to the incubation. (B) EMSA with nuclear extracts prepared from untransfected CHO cells and cells transfected with expression plasmids for Gal-GRWT, Gal-GRL501P, or Oct-2. The radiolabelled oligonucleotide employed contained a single Gal4 binding site and an octamer motif. Nuclear extracts were preincubated prior to the addition of the labelled probe and competitor DNAs. Incubation was continued for a further 20 min prior to PAGE. The individual combinations of nuclear extracts and unlabelled competitor oligonucleotides (added at a 100-fold molar excess) are summarized at the top of the panel.