Fig. 2. Transcriptional activation of NFB-dependent genes is not required for p65-mediated upregulation of the Hes1 promoter. NIH-3T3 cells were transfected with increasing amounts of IB_32-36 in the presence of N1-IC (0.5 µg) and p65 wt (0.5 µg). The reporter constructs were (A) 2xB-IL2-luc (1 µg) or (B) Hes 1-luc (1 µg). (C) Immunofluorescence assays to determine the subcellular localization of N1-IC and p65wt when coexpressed with IB_32-36 in NIH-3T3 cells. Cells were cotransfected with N1-IC, p65 wt and IB_32-36. N1-IC was detected with the 9E10 antibody and a FITC-labeled secondary antibody. p65 was detected with -p65 and a Cy3-conjugated secondary antibody. (D) Luciferase fold activation obtained by cotransfecting the indicated amounts p65TA with N1-IC (0.5 µg) and the Hes1-luc reporter (1 µg). Luciferase activity is presented as the fold induction relative to the basal level measured in cells transfected with empty vector. The average and standard deviation of duplicates from a representative experiment are presented. Lower panels show N1-IC and p65TA protein levels. Protein loading for each lane was normalized according to ß-galactosidase expression.