Immunosuppressive Drugs Affect High-Mannose/Hybrid N-Glycans on Human Allostimulated Leukocytes

Figure 2

Immunosuppressive drugs change the intensity of bands containing GNA-positive proteins. Peripheral blood mononuclear cells were cultured for 6 days in the presence of the combination of CsA (150 ng per 1 mL medium) and Rapa (12 ng per 1 mL medium) in a two-way mixed leukocyte reaction (MLR) and then lysed in RIPA buffer. Whole-cell lysate proteins (500 μg) were precipitated with 25 μL agarose-bound Galanthus nivalis lectin (GNA). The captured glycoproteins were recovered by boiling in Laemmli sample buffer with β-ME and resolved by 10% SDS-PAGE under reducing conditions. One-fifth of the GNA precipitate was destined for Western blotting (WB) and after probing with biotinylated-GNA was visualized by AP colorimetric reaction (a). The remaining four-fifths of the precipitate, after resolving on the same gel, were stained with Coomassie Brilliant Blue (CBB) and the bands were excised for MS/MS analysis (b). Molecular weight of proteins was assigned using a PageRuler Prestained Protein Ladder (Thermo Scientific, 26616). LP: lectin precipitation, C: untreated cells, CsA: cyclosporin A, and Rapa: rapamycin.

(a)

(b)