L. pneumophila infection induces NF-κB activation. (A) Time course of NF-κB activation in A549 and NCI-H292 cells infected with L. pneumophila, evaluated by EMSA. Nuclear extracts from A549 and NCI-H292 cells, infected with AA100jm strain (MOI of 100), for the indicated time periods, were mixed with either NF-κB (upper panels) or AP-1 32P-labeled probes (lower panels). Probes NF-κB and AP-1 are -83 to -68 bp and -130 to -116 bp fragments containing NF-κB and AP-1 binding sites of the IL-8 promoter, respectively. (B) Sequence specificity of NF-κB binding activity and characterization of NF-κB proteins that bound to the NF-κB binding site of the IL-8 gene. Competition assays were performed with nuclear extracts from A549 and NCI-H292 cells infected with AA100jm strain (MOI of 100) for 12 h. The competitor used was the NF-κB site of the IL-8 gene (lane 2). An unrelated AP-1 binding site was also used as a competitor (lane 3). Where indicated, 100-fold excess amounts of each specific competitor oligonucleotide (lanes 2 and 3) were added to the reaction mixture with labeled NF-κB probe. Specific bands are indicated by arrows. Supershift assay of NF-κB DNA-binding complexes in the same nuclear extracts was also performed. Gel shift assay reaction mixtures containing the same nuclear extracts and indicated antibodies were incubated for 45 min, and then 32P-labeled probes were added (lanes 4–8). Supershifted bands with anti-NF-κB p50, anti-NF-κB p65 and anti-c-Rel antibodies are indicated by arrowheads (lanes 4–6). (C) L. pneumophila infection leads to IκBα phosphorylation and degradation. A549 cells were infected with AA100jm strain (MOI of 100), for the indicated time periods. The cells were then lysed and analyzed by immunoblot with phospho-specific IκBα, IκBα and actin antibodies. Representative results of three similar experiments in each panel are shown.