S534 phosphorylation affects DNA binding and gene expression by NF-κB at late time points through regulation of p65 stability. S534 phosphorylation affects.

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S534 phosphorylation affects DNA binding and gene expression by NF-κB at late time points through regulation of p65 stability. S534 phosphorylation affects DNA binding and gene expression by NF-κB at late time points through regulation of p65 stability. (A and B) WT and S534A mice were injected intravenously with LPS (20 mg/kg) and sacrificed after the indicated times. (A) Liver tissue was analyzed by immunohistochemistry to monitor the translocation of p65 (red) to the nucleus (blue). (B) The percentages of cells with p65-positive nuclei were quantified. Data are means ± SD of five mice per genotype and time point. (C) Top: HEK 293 cells overexpressing human M2-p65 or M2-S536A-p65 were treated with TNF-α and then subjected to pulse-chase analysis for the indicated times to determine the half-life of p65 protein. Bottom: Data are means ± SD of three independent experiments, each performed in triplicate. AU, arbitrary units. (D) Top: WT and S534A MEFs were treated with cycloheximide (30 μg/ml) and then were left unstimulated or were stimulated with IL-1β for the indicated times. Samples were analyzed by Western blotting with antibodies against the indicated proteins. Bottom: The relative abundance of p65 protein, normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was determined for the indicated times by densitometric analysis. Data are means ± SD of three experiments. (E) Left: NF-κB DNA binding activity was determined in nuclear extracts from the livers of LPS-treated WT and S534A mice (n = 6 mice per genotype and time point). A value of 100 represents the positive control (Pos ctrl) (recombinant human p65). The dashed line indicates NF-κB binding activity at baseline. Right: The competitive oligonucleotide suppressed DNA binding by the positive control. *P < 0.05. Data are means ± SD of six mice per genotype. Jean-Philippe Pradère et al., Sci. Signal. 2016;9:ra85 ©2016 by American Association for the Advancement of Science