NF-κB signal is essential for flagellin-dependent activation of the IL-8 promoter by L. pneumophila. (A) Flagellin is required for induction of NF-κB binding activity. Nuclear extracts from Jurkat cells infected with Corby or flaA mutant were mixed with IL-8 NF-κB probe (MOI, 100:1). (B) Sequence specificity of NF-κB binding activity and characterization of proteins that bound to the NF-κB binding site. Competition assays were performed with nuclear extracts from cells infected with Corby for 2 h. 100-fold excess amounts of competitor were added (lanes 3 to 5). A supershift assay in the same nuclear extracts also was performed. Antibodies (Ab) were added (lanes 6 to 10). Arrows indicate specific complexes, while arrowheads indicate the DNA binding complexes supershifted. (C) Flagellin-induced p65 translocation. Cells were infected with Corby or flaA mutant. Nuclear extracts were subjected to immunoblotting. (D) Flagellin activates NF-κB through the classical and alternative pathways. Cells were infected with Corby or flaA mutant. Lysates were subjected to immunoblotting. (E) Overexpression of dominant negative mutants inhibits L. pneumophila-induced activation of the IL-8 promoter. Cells were transfected with -133-luc and the mutant plasmids and then infected with Corby for 6 h. The solid bar indicates luciferase activity of -133-luc and empty vector without infection. Activity is expressed relative to that of cells transfected with -133-luc with further Corby infection, which was defined as 100. Data are means ± SD values of three experiments. dn, dominant negative. *, P < 0.05; **, P < 0.001 (by Student t test).