Fig 2.

Inhibition of NF-κB by ectopic expression of wild-type or K173A mutant InlC. HeLa cells were transfected with plasmids expressing EGFP alone or EGFP-tagged wild-type (wt) InlC or InlC.K173A. About 24 h posttransfection, cells were stimulated for 30 min with 25 ng/ml TNF-α. (A) Effect of wild-type or K173A mutant InlC on nuclear translocation of NF-κB. After fixation and labeling of samples, confocal microscopy was used to analyze nuclear distribution of NF-κB in EGFP-expressing cells. (i) Representative images of HeLa cells expressing the various EGFP-tagged proteins. Note that expression of EGFP-InlCwt or EGFP-InlC.K173A each inhibited nuclear accumulation of NF-κB compared to expression of EGFP alone. (ii) Quantification of the effect of ectopic InlC expression on NF-κB nuclear localization. The fraction of NF-κB in the nucleus of cells expressing the various EGFP-tagged proteins was quantified as described in Materials and Methods. (B) Effect of wild-type or K173A mutant InlC on activity of an NF-κB-dependent reporter gene. Statistical analysis by ANOVA indicated that P was <0.0001. *, P < 0.05 relative to EGFP-expressing cells treated with TNF-α.