FIG. 6.

GR and AP-1 independently occupy a composite response element, consisting of an imperfect half-GRE and a neighboring AP-1 motif. (A) Sequences of 5′-biotinylated double-strand oligonucleotides used in the in vitro promoter complex assembly assay: WT, 40-bp Notch4 promoter oligonucleotides with wild-type GRE and AP-1 motifs; mut-AP1, 40-bp Notch4 promoter oligonucleotides with mutated AP-1 motif; mut-GRE, 40-bp Notch4 promoter oligonucleotides with mutated GRE site. The GRE and AP-1 motifs are indicated in uppercase boldface type. The mutated nucleotides are indicated in lowercase boldface type. (B) Western blotting analysis of proteins adsorbed to biotinylated oligonucleotides (representative pictures from at least three independent experiments). Monoclonal anti-GR antibody and polyclonal antibody against all Fos subunits were used for immunoblotting as indicated. The density of the specific bands was quantitated, and the value obtained for the WT condition was designated 1.0 (mean ± standard error of the mean for three independent experiments).