Fig. 1. The C/EBPβ-binding site is required for Id-1 expression. (A) Three C/EBPβ-binding sites are diagrammed as filled boxes, and their sequences shown on top. Mutations are indicated with lower case letters. The position of PBE (gray box) relative to Id-1 exons (hatched boxes) is illustrated. (B) EMSA with each of the three C/EBPβ-binding sites as probes. A 70 bp probe containing the β1, β2 or β3 site was incubated with Ba/F3 nuclear extracts with or without antibodies against C/EBPβ. The binding reactions were analyzed on a 6% poly acrylamide gel in Tris–glycine buffer. C/EBPβ-containing complexes are labeled as C1–C3. Supershifted complexes are marked with an open arrow. Additional bands are non-specific complexes found in all cell types. (C) Mutational analysis in transient transfection assays using Ba/F3 cells. Constructs containing the 460 bp PBE placed upstream of a luciferase reporter gene driven by a c-fos minimal promoter are diagrammed. Filled and open boxes represent wild-type and mutant C/EBPβ sites, respectively. Luciferase activities were normalized against the β-galactosidase activity. The data are presented as activities relative to that of the PBE-luc construct and are averages of at least three independent experiments with standard deviations. (D) Mutational analysis in stable Ba/F3 cell lines. The ΔId-1 gene is diagrammed as in (A). Three independent cell lines stably transfected with the indicated constructs were analyzed using RPAs, and transcripts were detected with the probes as labeled.