Figure 3.

The Egr-1 DNA binding site is required for PMA/PHA–responsive activation and for transcriptional synergy mediated by RelA and Egr-1. (A) CEM cells were transfected with either the wild-type NF-κB1 promoter (SSLUC) or with each of the site-directed mutants including (a) mNLUC, which contained a mutated NF-κB site at position −289, (b) mELUC, which contained a disrupted 68-bp Egr-1 site, and (c) mNELUC, which contained mutated NF-κB and Egr-1 sites, and 18 h later cells were stimulated with either PMA/PHA or TNF-α. 24 h after stimulation, cell extracts were harvested and assayed for luciferase activity. (B) CEM cells were co-transfected with either the SSLUC construct or with mutant reporters (mN, ME, mNE; 5 μg each) and with various expression vectors (5 μg) including an empty vector control, RelA, Egr-1, or RelA plus Egr-1. Cells were harvested 48 h after transfection and luciferase activities were determined. (C) The SSLUC construct was transfected into CEM cells along with either the vector control or with an Egr-1 expression construct. 18 h after transfection, cells were either left untreated or stimulated with PMA/PHA or TNF-α. Cells were harvested 24 h after stimulation and extracts were analyzed for luciferase activity. All transfection assays were performed in triplicate in three independent experiments.