TCR-stimulated Ras activation in Jurkat T cells and the effects of pharmacologic inhibitors.

Jurkat T cells were preincubated with herbimycin A (protein tyrosine kinase inhibitor), U73343 (inert analogue) or U73122 (PLC-γ1 inhibitor), and then stimulated with OKT3 at either a saturating concentration (10 μg/mL) or at a suboptimal concentration (0.156 μg/mL). Cell lysates were assayed for Ras-GTP using GST-Raf affinity selection followed by immunoblotting to detect precipitated Ras. Phospho-Erk and total Ras in lysates were also detected using immunoblot methods.