Figure 3.

CRAMP expression in IECs during postnatal development. (A) Quantitative analysis of CRAMP mRNA expression in primary IECs isolated from the small intestinal tissue of fetal, 6- and 24-h-, and 3-, 6-, 14-, 21-, and 28-d-old mice. Values represent the mean ± SD of gene expression determined in total IECs from three individual mice and indicate the target/housekeeping (Hprt1) gene expression ratio. n.d., not detectable. (B) Immunoblot for CRAMP in cell lysate of isolated IECs from 3- and 28-d-old mice (left) as well as in culture supernatant and cell pellet of isolated IECs from 3-d-old mice (right). Actin staining was included to demonstrate equal protein loading. (C) Immunostaining for CRAMP (red) in the small intestinal tissue of 3-, 6-, 14-, 21-, and 28-d-old mice. The FITC-conjugated lectin wheat germ agglutinin (WGA; green) binding to the mucus surface, and DAPI (blue) was used to delineate the anatomical structures of the mucoid surface and cell nuclei, respectively. Bar, 75 μm. (D) RT-PCR expression analysis for the known CRAMP-cleaving enzymes proteinase 3, pancreatic elastase, and kallikrein 5 and 7 in primary IECs of 1-, 6-, 14-, and 28-d-old mice, as well total small intestinal (SI) and skin tissue as positive control. H₂O and genomic DNA were included as negative controls for the intron-spanning primers.