LGALS3 protein, human

Fecal samples were prepared as previously described[22 ]. Briefly, 1g of fecal samples was diluted, mixed, and homogenized in 5mL of protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, United States; P83401)[22 ]. Blood was obtained from patients and healthy control subjects at 8 am and serums were separated, collected and stored at ﹣80 °C before use. Concentrations of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-17 (IL-17), and Gal-3 were measured in serum and fecal supernatants of UC patients by using commercially available ELISA tests, according to the manufacturer’s instructions.

Recombinant human galectin-3 and mutants and Xenopus galectin-3 were produced in E. coli BL21Star (DE3) cells (Invitrogen) and purified by affinity chromatography on lactosyl-Sepharose essentially as described for wild type human galectin-3 (34 (link)) but with some variation to optimize yield for each protein. The initial yields ranged from 3 mg/liter culture for G182A to 80 mg/liter for R144S, but, for example, lowering the temperature of the isopropyl 1-thio-β-d-galactopyranoside induction from 37 to 30 °C increased the yield of G182A galectin-3 about 10-fold. Mouse galectin-3 was produced from vector pIN III ompA2 in E. coli JA221 cells as previously described (36 (link)), except that Tryptone soy broth was used. The bacteria were processed and galectin-purified in the same way as for human galectin-3 described above. The galectins, in phosphate-buffered saline (118 mm NaCl, 67 mm Na⁺/K⁺-phosphate, pH 7.2) containing 4 mm β-mercaptoethanol, 2 mm EDTA and 150 mm lactose, were dialyzed against 2 liters of water that was changed once every 2 h 7 times and lyophilized and stored at −20 °C until use. This procedure does not completely deplete the galectin-bound lactose, which helps to preserve stability. Before use, the galectins were dissolved in the appropriate buffer, and any remaining lactose was removed by repeated ultrafiltration and concentration in a Centricon Plus-70, Ultracel PL10, or Centriprep Y10 ultrafiltration cell (Millipore AB, Sundbyberg, Sweden). Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad, Sundbyberg, Sweden). Protein size and integrity was determined by SDS-PAGE using 4–20% Precise^TM Protein Gels from Pierce (Nordic Biolabs, Täby, Sweden) in Tris/HEPES running buffer.
Differential scanning calorimetry measurements were performed on a MicroCal differential scanning calorimeter (MicroCal Inc., Northampton, MA) with a cell volume of 0.5072 ml. All samples were degassed for 15 min at room temperature before scanning. Protein samples (0.5 mg/ml) in PBS buffer with or without ligand were scanned in the temperature range 25 to 80 °C at a rate of 1 °C/min.The reversibility of the calorimetric traces was assessed by the reproducibility of scans upon rapid cooling to 25 °C followed by rescanning. Base-line scans were collected with buffer in both the reference and sample cells.

96-well plates were coated with recombinant human IgG1.Fc (A42561; Invitrogen) or human LRP-1 Cluster II Fc Chimera Protein covering ∼10% of the full-length Lpr1 protein sequence, including 3 N-glycosylation sites (#2368-L2-050; R&D Systems) at 0.5 μg in 100 μl PBS, incubated at 4°C overnight, and blocked with 50 mg/ml BSA for 90 min at 30°C. Serial concentrations ranging from 0.1 to 3.2 μg of recombinant human GALECTIN-3 (#774408; Biolegend), and test agents were added in a total volume of 50 μl and then incubated for 4 h at 30°C. Wells were washed, fixed with 2% PFA in PBS for 15 min at room temperature, washed, and incubated with rat anti-mouse monoclonal galectin-3 antibody (#125401; Biolegend; clone M3/38, epitopes mapped within the N-terminal region) for 30 min on ice. After washing, wells were incubated with AF488-conjugated donkey anti-rat secondary antibody (A-21208; Invitrogen Molecular Probes) for 30 min on ice. After washing, fluorescence was determined using a SpectraMax L (Molecular Devices) plate reader (excitation 485, emission 538) to quantify galectin-3–Lrp1 binding.

Recombinant human GALECTIN-3 protein (A13506; Abclonal) was incubated with activated recombinant MMP9 (RP00103; Abclonal) by adding 5 mM p-aminophenylmercuric acetate (A9563; Sigma-Aldrich) at 37°C for 30 min in 50 mM Tris-HCl buffer containing 150 mM NaCl and 10 mM CaCl₂ (pH 7.5). The incubation was stopped by adding with a volume of 4× Laemmli sample buffer, and the sample was separated on a 12% SDS-PAGE and analyzed by Western blot analysis using either anti–galectin-3 polyclonal antibody (rabbit anti-mouse antibody; 14979-1-AP; Proteintech) or anti–galectin-3 monoclonal antibody (mouse anti-mouse antibody; ab2785; Abcam).