NF-κB activation is increased in Raw 264.7 cells infected with the <i>iacP</i> mutant.

<p>(A) The expression of NF-κB p65 was determined using immunoblot analysis with an anti-NF-κB p65 antibody in the cytoplasmic and nuclear fractions of <i>Salmonella</i>-infected Raw 264.7 cells. The anti-GAPDH and anti-Lamin B antibodies were used as cytoplasmic and nuclear protein markers, respectively (Right panel). Quantification of the amount of nuclear and cytoplasmic p65 expression levels in Raw 264.7 macrophages. Bars correspond to means ± SD. (B) The nuclear localization of NF-κB p65 as monitored by indirect immunofluorescence was used as readout for NF-κB activation. The cell nuclei were stained with DAPI, and p65 was visualized with FITC-conjugated anti-p65 antibody (Right panel). Quantification of nuclear p65 activation in Raw 264.7 cells was performed by densitometry analysis. Images from five different confocal planes (at least 30 cells) per sample were analyzed to determine the quantification of nuclear NF-κB p65. The error bars represent the means ± SD for three individual experiments. Analysis by Student’s <i>t</i> test indicated that the differences were statistically significant (*, <i>P</i> < 0.05).