CD26-mediated redistribution of signaling molecules. (A) PBTCs and CBTCs (5 × 106) were left untreated or treated with 10 mM MβCD and incubated at 37°C for 5 minutes. Cells were stimulated via anti-CD26 mAb (1F7; 10 μg/mL) for 30 minutes. Samples were analyzed by SDS-PAGE under reducing conditions and Western blotting for antiphosphotyrosine mAb (4G10). Molecular weight markers are indicated. These results are representative of 10 different donors. (B) PBTCs and CBTCs (1 × 108) were lysed with alpha-methylnorepinephrine (MNE) buffer containing 1% Triton X-100, and the lysates were subjected to equilibrium gradient centrifugation. Electrophoresis of an aliquot of each fraction was performed under reducing conditions, and immunoblotting was performed with anti-Lck, anti-LAT, and anti-CD26 (5F8). (C) Distribution of phospho-Lck in PBTCs and CBTCs was analyzed with immunoblot for phosphotyrosine residues (4G10). The data are representative of 5 different donors.
