Figure 2.

ITLN1 mRNA and protein expression in the small intestine and colon. (a) ITLN1 mRNA expression in surgical specimens of ileum and colon according to diagnosis. The mRNA copy number per 10 ng total RNA was determined with quantitative real-time RT-PCR using external cDNA standards (see methods). (b) ITLN1 mRNA expression by V109D genotype (excluding ulcerative colitis samples). Box plots present the median and first and third quartiles, whiskers present the range. Statistical analysis of ITLN1 mRNA expression was performed using either a Kruskal–Wallis test with Dunn’s multiple comparisons or two-tailed Mann–Whitney U test; * p < 0.05. (c) Representative real-time bio-layer interferometry responses of recombinant ITLN1 V109 and D109 binding to immobilized β-D-galactofuranose (upper). Responses at 490 s were plotted against ITLN1concentration and fit to a single site binding equation to determine Kd (lower). Data is representative of three separate experiments. (d) SDS-PAGE (4–20% gradient) immunoblots of human ileum by V109D genotype (left, n = 2 per genotype) and disease state (right). (e) Immunohistochemistry of human colon using the following antibodies: ITLN conserved (ITLN^CONS), ITLN1-specific (ITLN1^NTERM), MUC2 mature (MUC2^C3), deglycosylated MUC2 (MUC2^APO, PH1900). Tissue counterstained by DAPI (blue). (f) Immunofluorescence of human colon using ITLN^CONS-FLOR (anti-chicken; Alexa Flour 647) and MUC2^C3 (anti-rabbit; Alexa Flour 488). (g) Immunohistochemistry of human ileum; ITLN2-specific (ITLN2^NTERM) antibody. Red box indicates Paneth cells identified with the ITLN^CONS antibody. CTRL = control (non-Crohn’s disease and non-ulcerative colitis), CD = Crohn’s disease, UC = ulcerative colitis. Images in (e–g) are representative of multiple (> 6 fields) from 9 specimens. Light microscopy scale bars: 20x (200 µm), 40x (100 µm), and 100x (50 µm); fluorescence microscopy scale bars: 20x (100 µm) and 63x (50 µm).