Renilla expression vector pgl4

To determine cell-density dependent Notch transcriptional activity, cells were cultured at either 1.5 × 10⁴ or 1.5 × 10⁵ cells in 1m l of medium/well. Cells were then incubated for 24 h and transfected with 1 μg of pGL3-CSL and 0.01 μg of Renilla expression vector pGL4.74 (Prom ega) in 1 ml of medium using Lipofectamine LTX reagent (Invitrogen). To determine Notch1 dependent IL-6 promoter activity, cells cultured at 8 × 104 cells/well were incubated for 24 h, transfected with 1 μg of either N1ICD or empty vector, IL-6 promoter reporter construct without or with point mutations in CSL binding site, and 0.01 μg of Renilla expression vector pGL4.74 (Prom ega) in 1 ml of medium using Lipofectamine LTX reagent (Invitrogen). After 48 h, the cells were harvested and the activity of firefly and Renilla luciferase was determined using the Dual Luciferase Assay System (Promega). The relative luciferase activity was calculated by normalizing firefly luciferase activity to that of Renilla luciferase to correct for the transfection efficiency.