Analysis of rTMD1 ligand by AlphaScreen, inhibitory effect of Le^y antigen on the binding of rTMD1 with LPS, and analysis of Lewis antigens in E coli LPS O111:B4. (A) AlphaScreen assay results. Values are the mean plus or minus SD (n = 4). Sugar binding specificity of mammalian-expressed rTMD1 is indicated by relative intensities (with reference to the highest absorbance unit). The sugar identities are designated by numbers as listed. ***P < .001 compared with the blank. The results were obtained from the average of 4 independent assays. Similar result was obtained using Pichia-expressed rTMD1. (B) Effect of Le^y antigen on the binding of rTMD1 with LPS. LPS was coated onto wells and various concentrations of Le^y and rTMD1 were added to each well. The binding of rTMD1 is expressed as the mean plus or minus SD (n = 4), *P < .05, **P < .01, and ***P < .001 compared with the rTMD1-only group. Similar results were obtained in 2 independent experiments. (C) Analysis of Lewis antigens in E coli LPS O111:B4. LPS (5 μg/well) was coated onto wells of high-binding microtiter plate. Le^a, Le^b, Le^x, and Le^y antibodies (5 μg/mL) were added to each well in binding buffer and incubated at 37°C for 2 hours followed by peroxidase-labeled secondary antibodies for 2 hours. The binding of Lewis antibodies to LPS was detected by measuring absorbance at 450 nm. Values are the mean plus or minus SD (n = 4). Similar results were obtained in 2 independent experiments. (D) Effect of Le^y on the blocking effect of rTMD1 on LPS-induced signaling pathways. rTMD1 (20 μg/mL) and various concentrations of Le^y were preincubated with LPS (100 ng/mL) before adding to cells. After incubation for 30 minutes, Western blot was used to assay LPS-induced ERK1/2 phosphorylation, degradation of IκBα in cytoplasmic fractions, and nuclear translocation of NF-κB p50 and p65 in nuclear fractions. Similar results were obtained in 2 independent experiments.