Goat anti galectin 3

Lgals3‐R200S wild‐type, heterozygous and mutant MEFs were harvested from 13‐day mouse embryos. Cells were cultured on 10 cm dishes until they reached 70% confluency. Experiments were carried out at 0–4 °C to reduce biotin internalization. Cells were washed three times with ice‐cold PBS pH 8.0 and labeled with 1 mg·mL⁻¹ EZ‐Link™ Sulfo‐NHS‐LC‐Biotin (Thermo Fisher Scientific) for 30 min with gentle rocking. The cells were then washed three times with ice‐cold PBS containing 100 mm glycine, and lysed in 50 mm sodium phosphate dibasic, 1 mm sodium pyrophosphate, 20 mm sodium fluoride, 2 mm EDTA, 2 mm EGTA, 1% Triton X‐100, 0.5 mm DTT, and complete protease inhibitor cocktail (Roche, Basel, Switzerland) lysis buffer. A portion of the whole cell lysate was kept as an input control. Remaining lysate was incubated with NeutrAvidin agarose beads (Thermo Fisher Scientific) for 3 h with slow rotation prior to washing three times with lysis buffer. Samples were boiled in loading buffer containing DTT prior to SDS/PAGE and western blot. Antibodies used were as follows: goat anti‐Galectin 3 (AF1197, R&D Systems, Minneapolis, MN, USA) diluted 1 : 10 000 and rabbit anti‐Vinculin (#4650, Cell Signaling Technology, Danvers, MA, USA) diluted 1 : 1000.

The solid-phase assay was performed in 96-well plates coated with Matrigel in PBS (5 µg/200 µL) overnight at 4 °C. Plates were blocked with 1% BSA for 1 h at 37 °C, and washed with PBS. Recombinant human CS-galectin-1, rhgalectin-3 and rhgalectin-8 at 0, 100, 500 or 1,000 ng/ml were added to the plates without or with I₄₇ (1,000 ng/ml), and incubated overnight at 4 °C. After washing with PBS, 100 µl of respective antibody for each galectin, rabbit polyclonal anti-galectin-1 IgG (Kirin Brewery, Japan), goat anti-galectin-3 (R&D) or goat anti-galectin-8 (R&D) were added and incubated for 2 h at room temperature (RT), with shaking. Wells were washed with PBS and incubated with biotinylated goat anti-rabbit IgG (for galectin-1) and horse anti-goat IgG (for galectin-3 and galectin-8). After 30 min, wells were washed, incubated with ABC for another 30 min and further with substrate and chromogen. The reaction was stopped with 0.2 M H₂SO₄. Non-specific binding was estimated by omitting the specific antibody.