Notch signaling induced by immobilized Delta4 ligand modulates EBF function. (A) RT-PCR analysis, visualized on an ethidium bromide-stained agarose gel, displaying the expression of Notch1 in the Baf/3 progenitor cell line and the 230-238 pre-B cell line. (B) Display of a Western blot analysis of HES-1 expression in BaF/3 cells after 36 hours of incubation on either the Fc-TRAIL-R4 or the Fc-Delta4 fusion protein, and after transfection of the cells with Notch1-IC as indicated. (C-D) Diagrams show the resulting relative luciferase activity obtained after DEAE-dextran-mediated transient transfections of 0.5 μg λ5 or CD19 promoter reporter constructs, 3.5 μg EBF, and 1.5 μg Notch1-IC, or 3.5 μg Pax-5 expression plasmids as indicated, into Baf/3 pro-B cells that then were incubated on plates coated with either the control protein Fc-TRAIL-R4 (▪) or Fc-Delta4 (□). The data are based on 3 transfections from a single representative of 3 experiments normalized to the internal control CMV Renilla. The reporter activities obtained with empty expression plasmid were set to 1 and error bars indicate SD. (E) Display of an EMSA where the DNA binding of ectopically expressed EBF to the Cd79a promoter EBF sites were assayed following incubation of the stably transduced cells on the control Fc-TRAIL-R4 or the Fc-Delta4 protein for 40 hours. (F) Diagrams show the expression of the endogenous λ5 gene in the cells stimulated in panel E. The data represent an average of 6 PCRs from 2 stimulation experiments and the error bars indicate the SD.