IKKβ(EE)^IEC mice exhibit persistent NF-κB activation, expression of NF-κB target genes, but no tissue damage. (a) Lysates of WT and IKKβ(EE)^IEC IECs were analyzed for villin and IKKβ expression by immunoblotting. Ln, lung; Sp, spleen; St, stomach; J, jejunum; I, ileum; C, colon. Representative of five experiments. (b) IKK activity was measured by immunocomplex kinase assay using GST-IκBα(1–79) as a substrate in lysates of WT and IKKβ(EE)^IEC jejunal (J), ileal (I), and colonic (C) enterocytes. Representative of five experiments. (c) Presence of the indicated proteins in cytoplasmic and nuclear extracts of WT and IKKβ(EE)^IEC enterocytes was examined by immunoblotting. Representative of five experiments. (d) Nuclear extracts of WT and IKKβ(EE)^IEC enterocytes were analyzed for p65 DNA binding by ELISA. Results (absorbance/micrograms protein) are means ± SEM (n = 3). *, P < 0.05 versus WT. Data are representative of three independent experiments. (e) WT or IKKβ(EE)^IEC enterocyte RNAs were analyzed in triplicates by Q-RT-PCR for expression of the indicated genes. mRNA amounts were normalized to Hprt mRNA. Results are means ± SEM (n = 4). *, P < 0.05 versus WT. Data are representative of four independent experiments. (f) ChIP was performed with a p65-specific antibody using fixed and sheared chromatin from WT and IKKβ(EE)^IEC small bowel enterocytes. Promoter sequences were analyzed by Q-RT-PCR. Results are means ± SEM (n = 3). *, P < 0.05 versus WT. Data are representative of three independent experiments. (g) WT and IKKβ(EE)^IEC enterocyte lysates (75 µg protein) were analyzed by ELISA for the indicated proteins. Results are means ± SEM (n = 3). *, P < 0.05 versus WT. Data are representative of four independent experiments. (h) Intestinal histology of WT and IKKβ(EE)^IEC mice. Representative of 10 experiments.