Figure 3

Overexpressing Par-4 reduces NFκB binding to Par-4 target gene promoters. (A) Transcription factor binding site analysis of Par-4-target genes for NFκB cis-acting sites. Response element sequence logos for NFκB family members p50 and p65 were generated from matrices in the TRANSFAC database using WebLogo (top panel) [76]. The height of each nucleotide base indicates overall conservation at that position. Representative sequence motifs identified by tffind for MAPK1, CDC27, CDK5, IGF1R, PDCD6 and TUBB genes are provided in the bottom panel. (B) ChIP-qPCR in HT29 cells transfected with empty vector pCB6+ or Par-4 expression vector. Chromatin DNA from p50- or p65-immunoprecipitates (ChIP), no antibody control (no Ab), or starting chromatin (Input) was amplified using quantitative PCR with primers for promoter regions of DROSHA, ITGB4, IGF1R, MT1X, MT1E, SLC2A1, MAPK1, CDK5, TUBB, or BRAF, and with primers for IL8 (positive control) and ACTB (nonspecific control). (C) and (D) PCR products were quantified by measuring 2^{(Ct Input - Ct ChIP)}, and the ratios of ChIP-to-input signals were used to yield relative NFκB p65 and p50 enrichment values. Averages and standard deviations from 6 independent ChIP experiments are plotted. *Significantly different NFκB occupancies at target genes between Par-4-overexpressing cells and empty vector-transfected cells (P < 0.05). See Additional file 2 for gene symbols and their corresponding gene names.