Figure 3.

Characterization of protein binding to the cis-regulatory elements on the aromatase promoter I.3/II region in LSMCs. EMSA was performed using oligonucleotide probes containing a C/EBP or CRE cis-regulatory element from the aromatase promoter I.3/II region, as indicated. All nuclear extracts (except the vehicle lane) were obtained from LSMCs treated with Bt₂cAMP (100 μm, 24 h). Unlabeled oligonucleotide was used as cold probe competitor, and competitors of mutated consensus sequences of the C/EBP binding site and CRE were used to confirm specificity of binding activity. Antibodies against C/EBPα, C/EBPβ, C/EBPδ, CREB-1, ATF-2, and CBP were used in supershift assays to identify proteins binding to each cis-regulatory element probe, and nonimmune IgG was used as a negative control. Distinct nuclear protein-DNA complexes occupied two C/EBP binding sites within the −317/−304 bp (A) and −245/−231 bp (B) probes. One of these complexes was supershifted upon addition of anti-C/EBPβ antibody and, although less prominently, anti-C/EBPδ antibody (arrow). Distinct nuclear protein-DNA complexes also occupied two CREs located at −292/−285 bp (C) and −211/−197 bp (D), but these complexes could not be supershifted with addition of any antibody tested. NE, nuclear extract; IgG, nonimmune rabbit IgG.