Supplementary Figures
Figure S1. Purification of secreted human colonic MUC2 mucin from LS 174T cells. (A) High
molecular weight glycoproteins (void volume V0 fractions 25-30) from the culture supernatants
(80% confluent cells) was fractionated on a Sepharose 4B column chromatography calibrated
with blue dextran (BD; Mr 2,000 kDa) and gel filtration standards bovine serum albumin (BSA;
Mr 67 kDa) and Vitamin B12 (B12; Mr 1.35 kDa). (B) Fraction of V0 mucin (2 mg) were
subjected to CsCl density gradient ultracentrifugation to purify MUC2 that partitioned in
fractions >1.46 g/mL. (C) CsCl mucin fractions 1-8 (500 ng/lane) were run on a 1.2% agarose
gel under reducing conditions and western blotted with an LS 174T MUC2 mucin antibody that
detected MUC2 mucins in fractions 6, 7 and 8. (D) CsCl fraction 7-8 (1μg) was run on a 1.2%
agarose gel and western blotted for MUC2, MUC5AC and MUC5B, demonstrating that the
purified mucin preparation contained primarily MUC2 with no contaminating MUC5AC or
MUC5B. (E) Silver stain of an SDS-PAGE (7.5%) gel of partially purified V0 mucin (1μg) from
Sepharose 4B column chromatography from (A) above and purified mucin in CsCl fraction 7
(1μg) from (B). There was no contaminating protein in the running gel in the CsCL fraction with
the majority of the poorly staining MUC2 mucin in the stacking gel as revealed by western blot.
Figure S2. (A) High MUC2 producing HT-29 cells shRNA silenced for MUC2 gene expression
demonstrating no concomitant alterations in MUC5AC and MUC5B gene expression. Gene
expression was quantified by qRT-PCR and represented as relative expression between high
producing MUC2 H and low producing MUC2 L cells. (B) hBD-2 peptide (200 pg) binding to
MUC2 mucin following incubation for 2 h as quantified by ELISA. Samples were triplicated
and experiments were replicated at least two times. One-way ANOVA/Post Tukey’s multiple
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comparison tests were run to determine significant differences among groups. (C) Confocal
immunolocalization of β-defensin peptides in the colonic mucosa of Muc2+/+ littermates with
antibodies against β-defensin 2 (yellow) at lower magnification. Nuclei were stained with DAPI
(blue). Image is from 1 of 5 independent experiments. Scale bar: 10 m.
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