Hrp labeling kit

Recombinant lectins (rHeltuba, rGal8N) were prepared using Escherichia coli as previously described (Tateno et al., 2011). Lectins were conjugated with HRP by using HRP labeling kit (Dojindo, Rockville, MD) at the concentration of 0.5 mg/ml and adjusted to the final concentration for incubation at 0.1 μg/ml.
One microgram of proteins from each cell population was separated by SDS‐PAGE on a 5‐20% gel (Perfect NT Gel system, NTH‐676HP, DRC, Tokyo, Japan) and transferred onto polyvinylidene fluoride membranes (Millipore, Burlington, MA). After blocking the membrane in Carbo‐Free blocking solution (Vector Laboratories, Burlingame, CA) for 1 hr at room temperature, it was incubated with HRP‐conjugated lectins overnight at 4°C. The signals were detected by using Western Lighting Plus (NEL104001EA, PerkinElmer). Lectin blot intensities were quantified using ImageJ software (National Institute of Health). The high‐intensity band was selected for quantification. Statistical significance was calculated by unpaired Student's t test (GraphPad Prism8 software).

SO and CHA powders were purchased from Sigma-Aldrich Co., LLC (Tokyo, Japan). Horseradish peroxidase (HRP)-labeled goat anti-rabbit polyclonal IgG (H + L) antibodies were obtained from Funakoshi Co., Ltd. (Tokyo, Japan). Commercial serum and urine samples of healthy volunteers were obtained from Cosmo Bio Co., Ltd. (Tokyo, Japan). Bio-Safe Coomassie stain, Clarity Western ECL substrate kits, Precision Plus Protein Dual Color Standards, Trans-Blot Transfer Packs, and 0.2 μm pore-size nitrocellulose membranes were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). A 96-well ELISA plate H was purchased from Sumitomo Bakelite Co., Ltd. (Tokyo, Japan). An HRP labeling kit was obtained from Dojindo Laboratories (Kumamoto, Japan). Blocking One and dimethyl sulfoxide (DMSO) were purchased from Nacalai Tesque, Inc (Kyoto, Japan). The Prominence LC-2010 HPLC system and the reversed-phase chromatography (RPC) column Shim-pack GIST C18-AQ were purchased from Shimadzu Corporation (Kyoto, Japan). Freund’s complete adjuvant (FCA), Freund’s incomplete adjuvant (FIA), o-phenylenediamine dihydrochloride (OPD) tablets, 2-mercaptoethanol (2-ME), and all other reagents were obtained from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan) and Wakenyaku Co., Ltd. (Kyoto, Japan).