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Figure 3 | Genome Medicine

Figure 3

From: High-throughput sequencing identifies STAT3 as the DNA-associated factor for p53-NF-κB-complex-dependent gene expression in human heart failure

Figure 3

p53 and NF-κB form a complex and occupy the putative GIS regulatory region simultaneously. (a) ChIP was performed on cardiac fibroblasts with or without DFX using antibodies against either p53 or RELA. Results show fold enrichment of real-time qPCR for the putative regulatory sequence (GIS). (b) ChIP using a p53 antibody was performed on cardiac fibroblasts with or without DFX and NFI. ChIP results are presented as mean ± standard error for three independent experiments performed in triplicate. (c) Using cell lysates from cardiac fibroblasts treated with DFX with or without NFI, RELA or control IgG immunoprecipitation (IP) was performed followed by western blotting (WB) for p53 (left), and vice versa (right). Arrows indicate RELA. (d) Cardiac fibroblasts were treated with or without DFX and NFI, and p53 ChIP was performed followed by 'release' of the chromatin, and RELA re-ChIP. Results represent fold enrichment of real-time qPCR for GIS. Re-ChIP results are presented as mean ± standard error for two independent experiments performed in triplicate. (e) Lysates from cardiac fibroblasts treated with or without DFX were incubated with streptavidin-coated beads on which biotinylated GIS duplexes (oligo pulldown) or scrambled sequence duplexes (scrambled) were immobilized. Proteins bound to these duplexes were eluted and western blotted for p53, RELA and NF-κB subunit p50. Asterisks represent P < 0.05 (paired t-test).

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