Sna biotin

Four hundred microliters of plasma were brought to a final volume of 0.5 ml in PBS, containing 1% NP40, 1% deoxycholic acid, and protease inhibitors. Then, 20 μl of 2 mg/ml SNA-biotin (Vector Laboratories, Burlingame, CA, USA) were added and the samples were stirred for 5 h at 4°C. Fifty microliters of streptavidin-agarose (Vector Laboratories) were then added and the samples were stirred overnight at 4°C. Samples were then centrifuged in a minifuge and washed three times with PBS containing 1% NP40, 1% deoxycholic acid, and protease inhibitors and once with 50 mM Tris-HCl, 15 mM NaCl. After centrifugation, samples were resuspended with reducing sample buffer, incubated at 60°C 15 minutes and electrophoresed.

For Western blots for ST6Gal1 protein, cells were lysed in RIPA buffer with added protease and phosphatase inhibitors. We used R&D Systems human ST6Gal1 antibody diluted 1:500 (Cat#AF5924) and β-actin as loading control (Santa Cruz, 1:500, Cat#sc-47778 HRP). We also assessed the effect of ST6Gal1 ablation on Lamp1 α2,6 sialylation. BCP-ALL cells were lysed in Triton T-100 lysis buffer with glycerol at pH 7.4 (Alfa Aesar, Cat#J63866AK) and glycoproteins were captured with SNA-biotin (Vector labs Cat #B-1305). Dynabeads Streptavidin magnetic beads (Invitrogen, Cat#65801D) were used to isolate the SNA-bound glycoproteins. Proteins were separated on 4%–20% SDS-PAA gradient gels (Mini-PROTEAN^® TGX Stain-Free™ Protein Gels, Bio-Rad, Cat#4568094). Lamp1 (CD109a) antibodies used at 1:1,000 dilution were from BioLegend (Cat#328602). The WB for OP9 cells used anti-Galectin-3 (BioLegend, 1:1,000, Cat#125402) or Galectin-1 (R&D Systems, 1:1,000, Cat#AF1152) antibodies. Western blotting for α2,6-sialylated proteins made use of biotinylated SNA from Vector Laboratories.

Lectin binding was evaluated by flow cytometry. The following reagents were used at the final concentrations indicated: SNA-biotin (Vector Laboratories B-1305, 20 μg/ml), MAL-II-biotin (Vector Laboratories B-1265, 10 μg/ml), allophycocyanin-streptavidin (APC-Strep) (Life Sciences, S-868), 5 μg/ml), LEA-biotin (Vector Laboratories B-1175, 1 μg/ml), L-PHA rhodamine (Vector Laboratories RL-1112, 20 μg/ml). K20 cells were cultured for the times indicated, with or without supplemental sugar, as indicated. Cells were harvested by centrifugation, washed twice with DPBS, then resuspended in DPBS at 2.0 × 10⁶ cells/ml. Then, 200 μl of cell suspension was transferred to V-bottom 96-well plate and pelleted by centrifugation. The cell pellets were incubated with 100 μl of lectin diluted in DPBS for 60 min at 4 °C (LEA incubation was 30 min) then washed with DPBS 3 times. When a secondary detection reagent was used, cells were incubated with 5 μg/ml allophycocyanin-streptavidin (Life Sciences, S-868) in DPBS for 45 min. Fluorescence was analyzed by flow cytometry on a FACSCalibur instrument (BD Biosciences) equipped with dual lasers at 488 nm and 635 nm. For sialidase experiments, before lectin staining, cells were incubated with Sialidase A (Prozyme GK80040) for 90 min at 37 °C in DPBS containing 0.1% BSA. Plots show the mean fluorescence intensity (MFI), typically for 10,000 cells.