<i>Capn8^CS/CS</i> mice are susceptible to ethanol-induced mucosal lesions.

(A) Schematic representation of the targeting vector and WT, CS-neo, and CS alleles of mouse Capn8. Exons 3 to 7 are indicated by black boxes with exon numbers. The 3′-probe for Southern blotting is shown as a box with hatched lines. The PCR primer positions for the genotyping of Cre-recombinant mice are shown by arrows. Neo, neomycin-resistance gene; DT-A, diphtheria toxin A fragment. (B) (left) Southern blot analysis of genomic DNA extracted from the tail of WT, Capn8CSneo/+ (CSneo/+), and Capn8CSneo/CSneo (CSneo/CSneo) mice. (right) PCR analysis of genomic DNA extracted from the tail of WT (+/+), Capn8CS/+ (CS/+), and Capn8CS/CS (CS/CS) mice. Intercrossing of heterozygous mice generated wild-type, heterozygous, and homozygous mice at a ratio not significantly different from the expected Mendelian ratio. M, DNA marker. (C) Western blot analysis of the gastric mucosal homogenates (20 µg) prepared from WT and Capn8CS/CS (CS/CS) mice. (D) The gastric mucosal homogenates from WT (lanes 1–4 and 9–12) and Capn8CS/CS (lanes 5–8 and 13–16) mice were incubated with or without Ca2+ and inhibitors, as indicated (CSTN, recombinant human calpastatin domain 1 fragment; E64, E64c). The samples were subjected to western blot analysis using an anti-calpain 8 (lanes 1–8) or anti-calpain 9 (lanes 9–16) antibody. Open arrowheads and asterisks indicate proteolytic fragments of calpain 9 and non-specific signals, respectively. (E) (left) WT and Capn8CS/CS mice were orally given 40% ethanol, and the lesion index was determined. Values are the means ± SEM. *, P<0.05 vs. WT. (right) Representative macroscopic views of the gastric mucosa of WT and Capn8CS/CS mice 4 hours after ethanol administration. Bars, 5 mm.