NF-κB binds to and augments Notch-dependent cyclin D3 promoter activity. (A) Cyclin D3 luciferase reporter plasmid (0.4 μg) and 0.1 μg pRL-CMV plasmid of internal control were transiently transfected into 293T cells. p50 and Notch1^IC expression plasmids were cotransfected in ratios, as indicated, together with the reporter plasmids. Notch1^IC was transfected in increasing amounts (0.1, 0.2, 0.3, and 0.4 μg) while the amount of p50 expression plasmid was kept constant (0.1 μg), and vice versa. Transfected cells were harvested 48 hours later for dual luciferase assay as previously described. Transfected cells were also harvested 48 hours later for whole cell lysates and immunoblotted for Notch-1 and p50. GAPDH was used as a loading control. (B) Cyclin D3 luciferase reporter plasmid was transiently cotransfected with either Notch1^IC expression plasmid or Notch1^IC mutants, as indicated, along with the p50 expression plasmid in 4:1 ratios (0.4 μg Notch1^IC:0.1 μg p50) into 293T cells. Transfected cells were harvested 48 hours later for dual luciferase assay. Values shown for both luciferase assays are representative of 3 separate experiments carried out in triplicate. (C) ChIP of stimulated CD4⁺ T cells with or without GSI treatment (as described in “Methods”). Anti-p50 was used to precipitate protein complexes bound to DNA. Goat isotype control IgG was used as a negative control. Two different primer sets (1 and 2) were used to assess specificity of binding of p50 to the cyclin D3 promoter via PCR of immunoprecipitated DNA fractions. Results shown are representative of at least 3 separate experiments.