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. 2012 Nov 23;41(2):764–774. doi: 10.1093/nar/gks1120

Figure 3.

Figure 3.

S329 phosphorylation reduces NF-κB binding to promoters with −1C-bearing κB-sites. (A–D) Quantitative ChIP assays using promoter specific primers for the TAP1/LMP2 or NOD2 promoters as indicated. IP was performed with anti-p50, anti-GFP or anti-histone H1 (positive control) as shown. Data represent promoter enrichment of p50, GFP or histone H1 relative to IgG control ± SEM of three separate experiments. (A and B) U87 cells were treated with 100 µM TMZ or vehicle for 16 h. (C and D) U87 cells were treated as in (A) following expression of the GFP-tagged p50 isoforms. *P < 0.05 relative to untreated p50wt samples. Inset: immunoblot with anti-p50 demonstrates equal expression of GFP-p50 mutants in stable clones. (E and F) Luciferase assays using a human NOD2 promoter/reporter construct with the indicated −1 nt mutation following treatment with TMZ for 16 h. (E) U87 cells were treated with the indicated TMZ concentrations. (F) p105−/− MEFs were co-transfected with either p50wt (wt) or p50S329A (S329A) as well as the indicated reporter construct and then treated 24 h later with vehicle or 100 µM TMZ. Data show mean relative luciferase, normalized to control conditions, ±SD of triplicate samples. *P < 0.05 relative to untreated. (G) Left: stable expression of Bcl-xL cDNA under the control of the wt (−1A) or mutant (−1C) NOD2 promoter in U87 glioma cells. Cells were treated with vehicle (U) or 100 µM TMZ (T) and IB performed with the indicated antibody. Right: colony formation assay in stable transfectants treated with TMZ. Cell lines include U87 cells expressing empty vector (U87) or Bcl-xL under the control of the wt (−1A) or the mutated (−1C) NOD2 promoter. Data show mean value from three independent experiments, each with three separate clones, ±SEM. *P<0.05.