Fig. 7. Inactivation of all the Rho GTPases inhibits CD82 cosignaling activity. (A,B) Jurkat cells (5x10⁶ cells) were cultured for 15 minutes at 37°C on antibody-coated or non-coated plates. After lysis in SDS sample buffer, SDS-PAGE analysis was performed. (A) An immunoblot with anti-phosphotyrosine antibodies 4G10. Left panel: cells treated with (lanes 2, 5 and 7) or without (lanes 1, 3, 4 and 6), 0.1 µg/ml Toxin B for two hours. Stimulation: lanes 1-2, no mAb; lane 3, suboptimal doses of anti-CD3 (OKT3: 0.5 µg/ml); lanes 4-5, optimal doses of anti-CD3 (OKT3: 20 µg/ml); lanes 6-7, anti-CD82 and suboptimal doses of anti-CD3 (C11: 50 µg/ml, OKT3: 0.5 µg/ml). Right panel: before stimulation Jurkat cells were transfected with 3 µg/ml GFP vector and 15 µg/ml of N17Rho GTPases or empty vector and sorted by their levels of GFP expression after 48 hours in culture. (B) After dehybridization, the same membrane was reprobed with anti-ZAP70 rabbit antiserum. (C) Jurkat cells (20x10⁶ cells), treated or not with 0.1 µg/ml toxin B for two hours, were cultured for 15 minutes at 37°C on antibody-coated plates. After solubilization in lysis buffer, 500 µg proteins of each sample were immuno-precipitated with anti-ZAP70 or anti-LAT rabbit antisera. Immunoprecipitates were resolved by 7.5% (for ZAP) or 12% (for LAT) SDS-PAGE and blotted with the anti-phosphotyrosine mAb 4G10. The data shown are representative of four independent experiments.