The requirement of c-JUN for PEA3 enrichment on the Notch-4 promoter. (A) MDA-MB-231 cells were cotransfected with pcDNA3.1-PEA3 and phMB vector alone or with phMB-TAM-67 expression plasmid. Lysates were subjected to chromatin immunoprecipitation using either PEA3-specific antibody or an isotype control IgG on the Notch-1 or Notch-4 promoter, respectively. PEA3 enrichment was measured by quantitative PCR and normalized to IgG control. In an independent experiment, cells were cotransfected with an AP-1 luciferase reporter, Renilla luciferase and phMB or phMB-TAM-67. Dual Firefly and Renilla luciferase assays were performed to measure AP-1 activity. Statistical comparisons were performed between phMB-vector and phMB-TAM-67 using a two-tailed, unpaired Student's t-test for two comparisons. (B) MDA-MB-231 cells were cotransfected with pcDNA3.1-PEA3 expression plasmid and control siRNA or c-JUN siRNA. Lysates were subjected either to chromatin immunoprecipitation and quantified as described above or to SDS-PAGE followed by Western blot analyses to detect c-JUN and actin (loading control). Statistical comparisons were performed between control siRNA and c-JUN siRNA using a two-tailed, unpaired Student's t-test for two comparisons. (C) PEA3 was immunoprecipitated from MDA-MB-231 lysates exogenously expressing PEA3 followed by Western blot analysis to detect PEA3 and c-JUN. The light chain of IgG was detected for loading purposes. The Western blots are representative of three independent experiments. Error bars represent standard deviations. The statistical significance of three or more experiments was determined by performing a two-tailed, unpaired Student's t-test for two comparisons.