FIG. 2.

Western blot assay of effects of flagellin and PAK ΔfliC on Calu-3 NF-κB signaling. Calu-3 cells were incubated in Ringer's solution and treated with flagellin (10⁻⁶ g/ml), PAK ΔfliC (10⁸ CFU/ml, MOI of 100), or flagellin plus PAK ΔfliC for the times shown, lysed, and then processed for immunoblot analysis of NF-κB p65, IκΒα, phospho-IκΒα (serine 32), IKKα, and phospho-IKKα/β (serine176/180). Binding of primary antibodies was visualized with horseradish peroxidase-conjugated secondary antibodies and quantitated (A to F) with NIH Image, normalized to maximal intensities measured during the experiment. See the text for details. The results shown are typical of three similar experiments.