Elevated TNF production in LPS-challenged IKKβ(EE)^IEC mice and mortality depend on MAPK activation. (a) Lysates from small intestinal enterocytes prepared 30 min after LPS challenge were analyzed for expression and phosphorylation of the indicated proteins by immunoblotting. Representative of three experiments. (b) Circulating TNF after LPS injection (0.2 mg/kg) with or without RWJ67657 (RWJ) or PD184352 (PD). Results are means ± SEM (n = 3). *, P < 0.05 versus IKKβ(EE)^IEC; #, P < 0.05 versus IKKβ(EE)^IEC with RWJ or PD. Data are representative of three independent experiments. (c) Small intestinal enterocyte lysates (75 µg protein) of indicated genotypes were prepared 90 min after LPS injection (0.2 mg/kg) with or without RWJ67657 (RWJ) or PD184352 (PD) and analyzed for TNF content by ELISA. Results are means ± SEM (n = 3). *, P < 0.05 versus IKKβ(EE)^IEC; #, P < 0.05 versus IKKβ(EE)^IEC with RWJ or PD. Data are representative of three independent experiments. (d) Rectal temperature and survival after LPS injection (0.2 mg/kg) with or without RWJ67657 (RWJ) or PD184352 (PD). Results are means ± SEM (n = 6). WT (gray diamond), IKKβ(EE)^IEC (black square), IKKβ(EE)^IEC + RWJ (black circle), IKKβ(EE)^IEC + PD (gray triangle), and IKKβ(EE)^IEC + RWJ + PD (black triangle). Survival of WT (gray diamond) and IKKβ(EE)^IEC + RWJ + PD (black triangle) is 100%, and survival of IKKβ(EE)^IEC + RWJ (black circle) and IKKβ(EE)^IEC + PD (gray triangle) is 100% up to 6 h. *, P < 0.05 for WT and IKKβ(EE)^IEC + RWJ + PD versus IKKβ(EE)^IEC; #, P < 0.05 for IKKβ(EE)^IEC versus IKKβ(EE)^IEC + RWJ or PD, and WT and IKKβ(EE)^IEC + RWJ + PD versus IKKβ(EE)^IEC + RWJ or PD. Data are pooled from two independent experiments. (e) The indicated mouse strains were analyzed 4 h after TNF administration (mice were injected i.v. with 2 µg of recombinant murine TNF). Small intestinal sections were stained with H&E. Arrows point at apoptotic cells. Representative of three experiments.