Lectin/western blotting

Biotinylated HPA (Sigma-Aldrich) was dissolved in 1× PBS containing 1mM CaCl₂ and 0.33 mM MgCl₂ to 1 mg/mL. Cells were lysed in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM CaCl₂, 1 mM MgCl₂ buffer supplemented with the protease inhibitor cocktail (Sigma-Aldrich) on ice for 1 h. Total protein concentration was estimated using BCA Kit (Pierce). Thirty micrograms of total cell lysate protein were resolved in 10% acrylamide gel by SDS-PAGE and then transferred onto the PVDF membrane. To avoid unspecific binding the membrane was incubated in a blocking buffer containing 5% BSA (Sigma-Aldrich) in TBST (TBS with 0.1% Tween-20 (Sigma-Aldrich)) for 3 h at RT. Next, the membrane was probed with 20 μg/mL HPA in 5% BSA, 1 mM CaCl2 in TBST solution for 1h followed by 3 × 10 minutes washing in TBST and incubation with HRP-conjugated streptavidin in 5% BSA, 1 mM CaCl₂ in TBST solution for 1 h at RT. After washing (5 × 10 minutes each) the positive signal was revealed using SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).

For the western blotting assay, membranes with transferred proteins were incubated for 1 h in 5% skimmed milk and probed with specific primary antibodies (Supplementary Table 2) overnight at 4°C. Secondary antibodies conjugated with HRP and Lumi-Light Western Blotting Substrate (Roche) were used to visualize specific protein bands. The bands were detected using Fujifilm LAS-3000 bioimaging and scientific research imaging equipment (FUJI Photo film Co., LTD.).