Lea biotin

Antibodies and concentrations used for immunoprecipitations are cataloged in Supplementary Table 2. Cells were lysed in 2% NP-40 buffer/Buffer A (150 mM NaCl, 0.5 mM Tris, 1 mM EDTA). Lysates were quantified using Bradford reagent. IGF1R, Integrins α4, β1, or β3 were immunoprecipated from at least 200 µg lysate mixed with 40 μl of BSA-blocked protein G-agarose bead slurry (#15920-010, Life Technologies) loaded with 2 µg anti-IGF1R (Abcam), anti-Integrins α4, β1 or β3 antibodies (Cell Signaling). Immunprecipitation was carried out overnight on a rotator at 4 °C, followed by washing with lysis buffer, elution by boiling in Laemmli reducing running buffer and western blot with LEA-biotin (Vector) lectin (for detection of I-branched glycans) or IGF1R, Integrins α4, β1 or β3 antibodies. As controls, immunprecipitations were performed in parallel with equal amounts of respective isotype control antibody.

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.