Figure 6. The function of RelA modifications on transcription and chromatin association is gene-specific.
(A), (B) and (F) Expression of the Vcam1 (A), Icam1 (B) and Mmp13 genes was examined further in the indicated by qRT-PCR. TNF stimulation for 1 or 8 h was used; error bars show standard deviations from three independent experiments. (C) Promoter-associated RelA ubiquitination is stimulated by TNF. Using U2OS cells with inducible replacement of endogenous ubiquitin by HA-tagged ubiquitin we performed a ChIP/re-ChIP experiment. Ubiquitin was immunoprecipitated next (using an HA antibody) and RelA was re-precipitated after elution. The human ICAM1 promoter region surrounding the κB site was detected by qPCR. The signal was normalized by the background precipitation (IgG control IP) and compared to the unstimulated sample (set as 1). (D), (E) and (G). RelA recruitment and histone acetylation at the Vcam1, Icam1 and Mmp13 promoters were determined by ChIP. As before, the respective DNA regions were detected by qPCR; the signal was normalized by the background precipitation (IgG control IP) and compared to the unstimulated sample (set as 1).