Constitutively active RBP-Jκ inhibits endothelial sprouting. (A) Expression of RBP-VP16 and N4IC in HMECs was detected by immunoblotting of total cellular lysates with a monoclonal anti-FLAG antibody and a monoclonal anti-HA antibody, respectively. Immunoblotting for tubulin demonstrates equivalent loading of total protein. (B) 4xRBP-Jκ luciferase reporter activity in HMECs transduced with RBP-VP16, N4IC, or empty vector control. Data are means + SD for a single experiment done in triplicate. Fold increases are reported for RBP-VP16 and N4IC cell lines as compared with the empty vector control. ^**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 by using single-stranded cDNA reverse-transcribed from total RNA isolated from HMECs transduced with RBP-VP16, N4IC, or empty vector control. PCR amplifications were done with primers specific for fragments of the HRT1-3 and GAPDH cDNA sequences. Amplification of the GAPDH fragment demonstrates equivalent levels of cDNA input. The relative patterns of mRNA expression are representative of at least 3 separate experiments. (D) Endothelial sprouting assay for HMECs transduced with RBP-VP16, N4IC, or empty vector control. Assays were quantitated and graphed as means + SE for an average of 4 experiments, each done in triplicate. ^*P < .05 and ^**P < .001 for the indicated comparisons.