Fig. 1

Identification of LINGO2 as a putative receptor for TFF3. a IL-10 secretion from the human macrophage cell line U937 in response to different rhTFF3 concentrations. Representative of two independent experiments. Means ± SE of quadruplicate wells are shown. b Mass spectrometry fold-enrichment peptide score following TRICEPS screen of U937 cells exposed to rhTFF3. c IL-10 produced by U937 following overnight treatment with rTFF3 in the presence of anti-human LINGO2 mAb. Means ± SE of quadruplicate wells with results of paired two-tailed t test are shown. Representative of two independent experiments. d Immunoprecipitation of LINGO2-Flag with either affinity purified TFF3-Fc or Fc only using protein A followed by immunoblotting with anti-Flag Ab (upper blot) or anti-IgG (lower blot). e Representative photomicrographs of HEK-293 cells single transfected with TFF3-RFP or f co-transfected with TFF3-RFP and LINGO2-GFP vectors, g co-transfected with TFF3-RFP and NH₂-terminal Flag-LINGO2 truncation mutant (Δ350 AA), h co-transfected with TFF3-RFP and CXCR4-GFP or (i) CXCR4-GFP and SDF-1-RFP. j Schematic of flow cytometry-based strategy to detect doubly fluorescent HEK cells following transfection with GFP labeled receptors and incubation with Alexa 647-labeled soluble protein ligands. k Representative flow cytometry dot plots showing the percentage of double positive cells following incubation of GFP-LINGO2 transfectants with PBS l IL-2-647, or m TFF3-647. n Double positive HEK following incubation of TLR2-GFP transfectants with TFF3-647, o with SDF-1-647 or p CXCR4-GFP transfectants with SDF-1-647. Data represent three independent experiments with mean ± SE and results of paired two-tailed t test shown. q Representative immunofluorescence images of rectosigmoid biopsy tissue samples from normal human subjects following co-staining with IgG isotype mAb or two different human subjects with anti-TFF3 mAb and anti-LINGO2 mAb (20×) (r, s). Scale bar represents 40 μm