FIG. 4.

Role of the NF-κB transcription factor in TLR2 promoter activity in A549 cells. (A) The TLR2 promoter construct pGL3-1486 was used as a wild type to produce the NF-κB deletion construct (pGL3-1486/NFm). The X indicates that the NF-κB transcription factor site has been deleted. Bars represent the fold induction by TNF-α, Dex, or both after 16 h. (B) The shortened TLR2 promoter construct, pGL3-297wt TLR2, was used as a template to construct pGL3-297/NFm, in which the 3′ NF-κB consensus site was deleted, as indicated. The bar graph represents the luciferase activity of the wild type (wt), mutated constructs, and pure pGL3-basic vector (empty vector) normalized to untreated cells. All samples were analyzed in duplicate, and the values are the mean ± SE of three experiments. (C) A549 cells were transiently cotransfected with the pGL3-297wt TLR2 reporter construct and the IκBα superrepressor expression plasmid that was ramped from 0 to 5 μg. The fold induction of luciferase activities by TNF-α, Dex, or both is shown. The luciferase activity of the pGL3-297 TLR2 construct in the presence of different concentrations of the IκBα superrepressor expression plasmid is shown. All samples were analyzed in duplicate, and the values are the mean ± SE of three experiments. *, P < 0.05 for pair comparison analysis (Tukey-Kramer test) to each control condition.