Notch4 induction of RBP-Jκ-dependent gene expression requires the ankyrin repeats. Reporter assays using a reporter construct with 4 copies of a RBP-Jκ-binding element upstream of an SV40 promoter-driven firefly luciferase gene (4xRBP-Jκ luciferase) (A) or a HRT2 promoter-driven firefly luciferase gene (HRT2 luciferase) (B). Reporter plasmids were electroporated into the HMEC-N4IC mutant cell lines along with a CMV promoter-driven renilla luciferase plasmid used as a normalization control for transfection efficiency. Cell lysates were harvested 48 hours after electroporation, and the relative luciferase units (RLUs) were determined as the ratio of firefly-derived luminescence over renilla-derived luminescence. Data are means + SD for a single experiment done in triplicate. Fold increases are reported for each N4IC construct cell line as compared with the empty vector control cell line. ^*P < .01 and ^**P < .001 for sample means compared with the empty vector control. The relative RLU patterns are representative of at least 3 separate experiments. (C) RT-PCR was performed using single-stranded cDNA reverse-transcribed from total RNA isolated from the HMEC-N4IC mutant cell lines. PCR amplifications were done with primers specific for fragments of the HRT1-3, HES1, HES4, and GAPDH cDNA sequences. Quantitation of 4 independent experiments was performed by densitometry of HRT2 (D) and HES4 (E) with levels normalized to GAPDH expression. ^*P < .05 and ^**P < .01 for sample means compared with the empty vector control.