Generation and molecular analysis of A20^IEC-KO mice. (A) Targeting scheme. The diagram shows the loxP-flanked (floxed) and deleted A20 alleles. The boxes indicate exons 1–9 (E1–E9). Restriction enzyme sites and the location of the probe used for Southern blot analysis are depicted. B, BamH1; V, EcoRV. LoxP and Frt sites are indicated by arrowheads. (B) Southern blot analysis on DNA from WT (+/+) and homologous recombinant (NFL/+) ES cells. (C) Western blot analysis for A20 expression in WT (+/+), heterozygous (+/−), and A20 knockout (−/−) primary MEFs either stimulated or not for 5 h with recombinant mouse TNF. (D) DNA isolated from various tissues of a A20^FL/FL/Villin-Cre⁺ mouse and a control littermate (A20^FL/FL/Villin-Cre⁻) was subjected to Southern blot analysis. Δ, deleted allele; FL, floxed allele; SI, small intestine. (E) Quantitative PCR measurement of A20 mRNA expression in purified IECs from A20^IEC-KO (n = 2) and control WT littermate mice (n = 2) 0 or 30 min after mouse TNF injection. Error bars represent SEM. (F) Western blot analysis for A20 expression in colonic epithelial cells from two individual A20^IEC-KO and control WT littermate mice. *, unspecific. Data are representative of two independent experiments.