PEA3 positively regulates Notch-1 and Notch-4 transcription. MDA-MB-231 cells were transfected with one of two unrelated scrambled siRNA (SCRBia or SCRBib) or one of two unrelated PEA3 siRNA (PEA3ia or PEA3b). (A) Relative mRNA levels of Notch receptors (NOTCH-1, NOTCH-2, NOTCH-3 and NOTCH-4) were measured by quantitative PCR. Relative mRNA levels of PEA3 were measured as knocked-down controls using quantitative PCR. (B) Protein levels of Notch-1 and Notch-4 were detected by Western blot analysis. For Notch-4 Western blot analysis, cells were treated with or without lactacystin for 24 hours. The Western blots are representative of three independent experiments. (C) Downstream targets of PEA3 (MMP-9) and Notch (HEY-1) were measured by quantitative PCR. Transcripts were normalized to HPRT, and mean values were plotted. 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.