Kinetics of LAT tyrosine phosphorylation in Jurkat T cells.

Wild-type Jurkat T cells (1 × 10⁶ cells/test) were either left unstimulated, stimulated with the mitogenic pair of anti-CD2 mAbs T11₂ and T11_3, or stimulated with anti-CD3ε mAb OKT3 at 37°C for the indicated periods of time. Cells were then lysed in 1% Brij97 lysis buffer, and 30 μL of clarified postnuclear lysates (PNL) (0.3 × 10⁶ cell equivalents) were resolved on SDS-PAGE, transferred to PVDF membranes, and immunoblotted with the antiphosphotyrosine antibody 4G10 (left panel). Note the kinetics of pp36-38 phosphorylation, a phosphoprotein identified as LAT by stripping the membrane and reblotting with anti-LAT–specific antibody (right panel, lower). Because the anti-LAT antibody less efficiently recognizes the phosphorylated form of the LAT protein, anti-PLCγ-1 and anti-Vav antibodies were used to confirm loading of equivalent amounts of proteins at different time points following stimulation (right panel, upper).