Ectopic expression of Notch1-IC impairs the DNA-binding ability of EBF. (A) EBF protein levels displayed on a Western blot obtained with an anti-c-myc antibody and 10 μL of the nuclear extracts also used for the EMSA (B) or nuclear extract from HeLa cells transfected with empty vector (MD). (B) An EMSA analysis where an end-labeled Cd79a promoter EBF-binding site was incubated with 6, 4, or 2 μL nuclear extract from HeLa cells transfected either solely with EBF or with EBF and Notch1-IC-encoding plasmids collectively. For nuclear extract quality control, 1 μL of the respective nuclear extract was incubated with an Oct1 protein-binding probe. (C) A schematic drawing of the EBF protein^28,29  with the DNA-binding domain indicated in white, an IPT/TIG (immunoglobulin-like, plexins, transcription factors/transcription factor immunoglobulin domain) domain by stripes, the dimerization domain by black, and the major transactivation domain substituted with that of the Vp16 protein by dots. (D) Diagrams display the relative luciferase activity of the λ5 reporter or a Gal-4 (G5) responsive reporter plasmid after transfection into HeLa cells in the presence or absence of EBFVp16 or Gal-4Vp16 protein and Notch1-IC-expressing plasmids. The data are based on a single representative of 3 experiments including 3 transfections normalized to the internal control pRL-0. The reporter activities obtained with empty expression plasmid were set to 1 and error bars indicate SD.