FIG. 11.

SAg induces PLCγ1 and Erk phosphorylation in P116 T cells. (A) Parental E6.1 Jurkat and ZAP-70-negative P116 T cells were stimulated at 37°C for 2 min by APCs loaded with the indicated amount of SEE. Cell lysates (10,000,000 T-cell and 2,000,000 LG2 cell equivalents) were subjected to immunoprecipitation with anti-PLCγ-1, transferred to a polyvinylidene difluoride membrane, and immunoblotted for phosphotyrosine (top). The membrane was then stripped and blotted for PLCγ1 (middle). Lanes: APCs; PLCγ-1 immunoprecipitate from 2,000,000 LG2 cell equivalents; Beads, immunoprecipitating antibody-coated beads alone without cell lysate. WCL proteins (450,000 T-cell and 90,000 LG2 cell equivalents) were resolved by electrophoresis on 8% polyacrylamide gels, transferred to a polyvinylidene difluoride membrane, and immunoblotted for dually phosphorylated, activated Erk (bottom). Lane APCs, WCL from LG2 cells (90,000 cell equivalents) alone. (B) Parental Jurkat and ZAP-70-negative P116 T cells were stimulated with APCs loaded with 300 ng of SEE per ml at 37°C for 0.5, 2, 5, 10, or 30 min. Tyrosine phosphorylation of PLCγ1 and activation of Erk in the cells were measured as in panel A. The membrane loaded with WCLs was also immunoblotted for ZAP-70.