LAT is tyrosine phosphorylated and associates with PLCγ-1, Grb-2, and other tyrosine phosphorylated proteins on CD2 stimulation.

Wild-type Jurkat T cells (10⁷ cells) were stimulated with either the mitogenic pair of anti-CD2 mAbs T11₂ and T11₃ or anti-CD3ε mAb OKT3 at 37°C for the indicated periods of time. PNLs were prepared as described in “Materials and methods,” either subjected to immunoprecipitation with the rabbit polyclonal antibody anti-LAT or incubated with protein A–agarose alone (not shown), separated by SDS-PAGE, and immunoblotted with the antiphosphotyrosine antibody 4G10 (A, upper panel). A longer exposure of the upper part of the same membrane is shown to demonstrate bands corresponding to phosphotyrosine-containing proteins of molecular weight between 70 and 203 kd (A, lower panel); in particular, phosphotyrosyl bands of approximate molecular weight of 120, 97, and 76 kd are indicated by open arrowheads. The membrane was stripped and reblotted with anti-LAT (B, upper panel), anti-Grb-2 (B, middle panel) and anti-PLCγ-1 antibody (B, lower panel). Results are representative of 3 independent experiments.