Constitutive p40 promoter activation and IL-23 production in the terminal ileum mediated by dendritic cells
J. Clin. Invest. Christoph Becker, et al. 112:693 doi:10.1172/JCI17464 [Go to this article.]

Figure 7
Binding of NF-κB to the IL-12 p40 promoter is upregulated in the distal small intestine. (a) EMSA analysis: 40 μg of tissue lysate from the small intestine (D1–D4) of FVB mice was incubated with a radiolabeled probe corresponding to the IL-12 p40 promoter NF-κB site. Protein/DNA complexes were analyzed on a 5% native polyacrylamide gel. Two representative experiments out of four are shown. (b) For supershift analysis, D4 protein lysate was preincubated with 2 μg of Ab’s specific for the indicated transcription factors prior to the addition of radiolabeled probe. The locations of the p50/p65 complex and the p50 supershift are indicated. (c) Constitutive luciferase activity in samples D1 (proximal) to D4 (distal) of the small bowel and spleen of IL-12 p40 wild-type promoter/transgenic mice and NF-κB mutant promoter/luciferase transgenic mice. Luciferase expression was measured in a standard luminometer after homogenization of organ samples of transgenic mice. Results were normalized to the protein content of the homogenate and are presented as relative light units per milligram of protein extract ± SD of three independent experiments with independent founder mice. A striking reduction of luciferase activity was noted in the distal small intestine of mice carrying a loss-of-function mutation in the NF-κB–binding site of the IL-12 p40 promoter as compared with transgenic mice carrying the wild-type p40 promoter upstream of the luciferase gene. In contrast, no reduction of luciferase activity in spleen cell lysates was noted.