FIG. 4.

SLP-76 and LAT are not required for Erk activation in CD3-stimulated P116 cells. (A) Jurkat and P116 T cells were stimulated by CD3 cross-linking at 37°C with 10 μg of UCHT1 per ml for 0, 0.25, 0.75, 2, 8, 15, or 30 min. WCLs were precleared with 20 μg of GST bound to glutathione-agarose. Grb2-associating proteins were precipitated from the cleared lysates with 20 μg of glutathione-agarose-immobilized GST-Grb2. The extent of tyrosine phosphorylation of associated proteins was determined by antiphosphotyrosine (4G10) immunoblotting. Shown are the 76- and 36-kDa proteins, which correspond to SLP-76 and LAT. (B) SLP-76-deficient (J14-v-29) and SLP-76-reconstituted (J14-76-11) Jurkat T-cell lines were stimulated with OKT3 ascites (1:50) for 0, 0.25, 0.75, 2, 8, 15, or 30 min at 37°C, and Erk activation was measured as described in the legend to Fig. 1. (C) LAT-deficient (JCaM2.5) and LAT-reconstituted (JCaM2.5.B3) Jurkat T-cell lines were analyzed as in panel B.