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

Figure 3

Rapamycin (Rapa) and the combination of cyclosporin A (CsA) and Rapa changes β1 and β3 integrin expression on MLR-activated cells. Peripheral blood mononuclear cells were cultured for 6 days in the presence of CsA (200 ng per 1 mL medium), Rapa (20 ng per 1 mL medium), and 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. Protein extracts (15 μg) were digested with endo-β-N-acetylglucosaminidase H (Endo H) from Streptomyces plicatus, SDS-PAGE-separated on 10% gel under reducing conditions, electroblotted onto a PVDF membrane, and probed with specific primary antibodies: mouse monoclonal anti-β1 (Chemicon, MAB2251, clone B3B11) and rabbit polyclonal anti-β3 (Chemicon, AB1932). Antibody-bound integrins were visualized by chemiluminescence. GAPDH was the endogenous control. C: untreated cells.