Fig. 1. CD82 engagement triggers tyrosine phosphorylation of Vav and SLP76. Jurkat cells were cultured for 10 minutes at 37°C on uncoated plates or plates coated with anti-CD82 (C11, 50 µg/ml). (A) upper panel, after lysis (5x10⁶ cells) in SDS sample buffer and SDS-PAGE analysis, tyrosine-phosphorylated proteins were analyzed by immunoblotting with 4G10 antibody. Stimulation, lane 1; no mAb, lane 2; anti-CD3_o (optimal OKT3, 20 µg/ml); anti-CD82 (C11, 50 µg/ml). (A) Middle and lower panels, after dehybridization, the same membrane was re-probed with anti-Vav1 rabbit (middle panel) or anti-SLP76 sheep antisera (lower panel). (B) After solubilization (20x10⁶ cells) in lysis buffer, 500 µg of each samples were immunoprecipitated with anti-Vav1 (rabbit polyclonal IgG), anti-SLP76 (sheep polyclonal IgG.) or control antibodies (C1, rabbit IgG; C2, sheep IgG). Immunoprecipitates were resolved by 7.5% SDS-PAGE and blotted with the anti-phosphotyrosine mAb 4G10 (upper panel), monoclonal mouse anti-Vav1 (middle panel) or polyclonal sheep anti-SLP76 (lower panel). The data shown are representative of three independent experiments.