NF-κB and GAS elements are important for LPS-induced activation of the CD40 promoter. (A) Deletion constructs of the human CD40 promoter. (B) RAW264.7 cells were transiently transfected with 0.2 μg of the indicated constructs, then treated with medium or LPS (10 ng/mL) for 12 hours and analyzed for luciferase activity. Values were normalized to total protein, and fold induction was calculated by dividing the LPS treatment values by UN levels. Data are presented as mean ± SD of 3 experiments. (C) Site-directed mutant constructs of NF-κB and GAS elements in the human CD40 promoter. (D) RAW264.7 cells were transiently transfected with 0.2 μg of the indicated constructs, allowed to recover for 4 hours, then were treated with medium or LPS (10 ng/mL) for 12 hours and analyzed for luciferase activity. Fold induction was calculated and presented as mean ± SD of 3 experiments. (E) RAW264.7 cells were transiently cotransfected with the WT CD40 promoter construct (0.2 μg) and expression vectors containing WT or double negative (DN) of IKK-α or IKK-β cDNA (0.1 μg), then they were treated with LPS (10 ng/mL) for 12 hours and analyzed for luciferase activity. Differences in the amount of DNA were adjusted with the empty vector pcDNA3. Data are presented as mean ± SD of 3 experiments.