SLAP associates with ZAP-70, Syk, LAT, and CD3ζ upon TCR activation. (A) GST-SLAP was transiently transfected into Jurkat T cells. Fifteen hours after transfection, cells were stimulated by OKT3.14 and RAM antibody at 37°C for 2 min. Cell lysates were incubated with glutathione-Sepharose beads. Bound proteins were separated by 10% SDS/PAGE and blotted with anti-p-Tyr or anti-GST antibody. Potential positions of Syk, ZAP-70, GST-SLAP, LAT, and CD3ζ chain are noted. (B) GST-SLAP-transfected Jurkat T cells were activated by OKT3.14 and RAM antibody. Cell lysates were incubated with glutathione-Sepharose beads, and precipitated proteins were blotted with anti-Syk antibody (lanes 1 and 2). Anti-Syk immunoprecipitations of Jurkat T cell lysates were used as a marker for the size of Syk protein (lanes 3 and 4). (C) Same as B, except anti-ZAP-70 antibody was used for immunoprecipitation and blotting. (D) Same as B, except anti-LAT antibody was used for immunoprecipitation and blotting. (E) Same as B, except anti-CD3ζ antibody was used for immunoprecipitation and blotting.