FIGURE 5.

Effects of c-Rel and RelA silencing on NF-κB subunit mRNA and protein levels. Forty-eight h after transfection of PC12 cells with control, c-Rel, or RelA siRNA, c-Rel and RelA expression was analyzed. A and B, c-Rel (A) and RelA (B) gene expression was quantified by real-time PCR. Data are expressed as mean ± S.E. (error bars) relative to control values (n = 4). *, p < 0.05 (Mann-Whitney U test). The experiment was repeated three times with similar results. C and D, immunocytochemistry was performed using antibodies directed against c-Rel (C) and Rel-A (D) subunits followed by a fluorochrome-conjugated secondary antibody. Nuclei were labeled with DAPI. Data are expressed as mean ± S.E. (error bars) relative to control values (n >30). ***, p < 0.001 (Student's t test). The experiment was repeated three times with similar results. E, cytoplasmic and nuclear fluorescence of c-Rel and RelA was measured using ImageJ software. Western blotting of RelA after siRNA treatment. α-Tubulin was used as a loading control.