Figure 8.

E-protein activity controls NF-κB activation. (a and b) 16610D9 cells were transiently transfected with NF-κB luciferase reporter (PGL4.32) and CMV-Renilla along with E47 and Id protein expression plasmids individually (a) or in combination (b). Transfected cells were cultured overnight, and then stimulated with 2 µg/ml anti-CD3 and 1 µg/ml anti-CD28 for 2 h. Luciferase activity was measured and normalized by Renilla luciferase activity. Data shown are derived from mean ± SD of duplicate transfections in each experiment and are representative of at least three independent experiments. (c) 16610D9 cells were retrovirally transduced with empty vector, Id1-, Id2- or Id3-expressing vectors, and were stimulated with 2 µg/ml plate-bound anti-CD3 and anti-CD28 for 2h. Whole-cell lysates obtained were immunoblotted with antibodies against the indicated proteins. Data are representative of three independent experiments.