TCR complex signal transduction, but not IL-2, is inhibited by dasatinib. T cells were stimulated in the presence or absence of dasatinib for 2 or 10 minutes at 37°C, then immediately lysed. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with (A) pan antiphosphotyrosine or (B) PathScan Multiplex Western Cocktail I (phospho-p90RSK, phospho-AKT, phospho-ERK, phospho-S6, and loading control eIF4E). Chemiluminescent detection was performed with a FluorChem SP CCD camera. (C) T cells were stimulated for 15 minutes with IL-2 and/or 5 minutes with OKT3, then fixed with formaldehyde at 37°C for 10 minutes. Cells were then permeabilized with methanol and stained for phospho-ERK or phospho-STAT5. Solid lines, stimulated T cells (10 μg/mL OKT3 alone or with 20 ng/mL IL-2 as indicated); dashed lines, stimulated T cells in the presence of 10 nM dasatinib; dotted lines, control. (D) T cells were stimulated with OKT3 or IL-2 for 30 minutes at 37°C, nuclear extracts were isolated, and DNA binding of STAT5 and NFκB was determined by EMSA.