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 buy GDC-0973 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 Idasanutlin in vitro 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). As described above, the flaA mutant strain failed to induce mRNA expression and production of IL-8. Next, we determined whether the flaA mutant strain induces NF-κB DNA binding activity. As expected, NF-κB DNA binding activity was not induced by the isogenic flaA mutant, unlike the wild-type strain Corby (Fig.

6A). These results indicate that better activation Cell press of NF-κB binding by flaA-positive strain is the underlying mechanism of the observed activation of the IL-8 promoter by this bacterial strain. Considered together, these results indicate that L. pneumophila infection induces IL-8 gene expression at least in part through the induced binding of p50 and p65 NF-κB family members to the NF-κB element of the IL-8 promoter and that this effect is dependent on flagellin. Because nuclear translocation is a key step for transcriptional activity [9], we next examined whether L. pneumophila induces the nuclear translocation of NF-κB. As shown in Fig. 6C, the wild-type Corby, but not the flaA mutant, induced nuclear translocation of NF-κB. NF-κB is normally present in the cytoplasm in an inactive state and is bound to members of the IκB inhibitor protein family, chiefly IκBα. In this complex, IκBα blocks the nuclear localization signal, thus preventing nuclear translocation. Translocation of NF-κB into the nucleus requires disruption of the cytoplasmic NF-κB:IκBα complex [9]. To determine the role of IκBα phosphorylation and degradation in L. pneumophila-induced NF-κB translocation and activation, we investigated whether L. pneumophila induces phosphorylation and degradation of IκBα.