PEA3 is enriched on the Notch-1 and Notch-4 promoters. (A) MDA-MB-231 cells were transfected with pcDNA3.1 alone or pcDNA3.1-PEA3 expression plasmid. Lysates were immunoprecipitated (I.P.) with IgG control or PEA3-specific antibody. Western blot analysis was performed to detect PEA3. Heavy and light chains of IgG were used for loading. The Western blots are representative of three independent experiments. (B) and (C) MDA-MB-231 cells transfected with pcDNA3.1 or pcDNA3.1-PEA3 expression plasmid were lysed, and lysates were subjected to chromatin immunoprecipitation using either PEA3-specific antibody or an isotype control IgG on the Notch-1 promoter (B) or the Notch-4 promoter (C), respectively. PEA3 enrichment was measured by quantitative PCR and normalized to pcDNA3.1 and IgG control. Schematic representations of Notch-1 and Notch-4 promoter regions and the primers used in the studies flanking the specific sites are shown. Error bars represent standard deviations. Statistical significance of three or more experiments was determined by performing a two-tailed, unpaired Student's t-test for two comparisons.