Silver stain kit

Proteins were resolved by 10 % polyacrylamide gel electrophoresis (PAGE) under reducing conditions and visualized by silver staining, performed using the Silver Stain Kit (BioRad, Hercules, CA, USA) according to the manufacturer's instructions. Proteins separated on 10 % polyacrylamide gel were transferred to nitrocellulose membrane by Western blotting. Non-specific binding was blocked by TBS (50 mM Tris-HCl, 150 mM NaCl) pH 7.6/1 % BSA for 1 h at room temperature. The membranes were incubated overnight at 4 °C with rabbit anti-human galectin-1 (1:3000, INEP) and goat anti-mouse galectin-1 affinity-purified polyclonal antibody (1:1000, R&D, Minneapolis, MN, USA), followed by secondary antibody-HRP (biotin-labeled goat anti-rabbit IgG, Vector Laboratories, 1:3000; Rabbit anti-Goat IgG (H+L), HRP, Invitrogen, Waltham, MA, USA, 1:10000). Non-specific binding was assessed by incubation with secondary antibody only. Protein bands were detected by ABC (in case of biotinylated secondary antibody) or by adding 0.05 % 3.3'-diaminobenzidine tetrahydrochloride (DAB) (Vector Laboratories).

Proteins were resolved on 10% separating gel with 4% stacking gel under denaturing and reducing conditions (27 (link)) and stained with silver nitrate, using a silver stain kit (Bio-Rad) according to the manufacturer’s instructions. The gel was calibrated with SDS-PAGE molecular weight standards (broad range).

Monoclonal anti-CD63 antibody (clone TS63) was from Abcam (Cambridge, UK) and biotinylated goat anti-galectin-3 (gal-3) antibodies from R&D Systems (Minneapolis, MN, USA); 3,3′,5,5′-tetramethylbenzidine (TMB), bovine serum albumin (BSA), D-lactose, and methyl- alpha D-mannopyranoside were from Sigma (St. Louis, MO, USA). Biotinylated goat anti-mouse IgG, biotinylated plant lectins, concanavalin A (ConA), SNA (Sambucus nigra agglutinin), and the Elite Vectastain ABC kit were from Vector Laboratories (Burlingame, CA, USA). Sephadex G 200 and Sephadex DEAE A-50 were from Pharmacia AB, Uppsala, Sweden. The silver stain kit and SDS-PAGE molecular mass standards (broad range) were from Bio-Rad (Hercules, CA, USA). Nitrocellulose membrane and Pierce ECL Western Blotting Substrate were from Thermo Scientific (Rockford, IL, USA). BCA Protein Quantification Kit was from Abcam (Cambridge, UK). Microwell plates were from Thermo Scientific (Roskilde, Denmark). Lymphocyte separation medium LSM-B was from Capricorn Scientific GmbH (Ebsdorfergrund, Germany).