RosA inhibits the TCR-induced tyrosine phosphorylation and subsequent activation of Itk.

(A) RosA inhibits TCR-induced tyrosine phosphorylation of Itk. Jurkat cells were preincubated with the indicated concentrations of RosA for 4 hours and stimulated for 3 minutes as described in “Materials and methods.” Supernatants of TCR-stimulated Jurkat cell lysates (5 × 10⁷) were precleared with mouse serum agarose and immunoprecipitated with anti-Itk mAb (5 μg/mL). Immunoprecipitates were resolved by 12% SDS-PAGE, and the blot was subsequently probed with antiphosphotyrosine mAb (0.5 μg/mL) or rabbit polyclonal anti-Itk antibody (1 μg/mL). Representative data from more than 2 similar experiments are shown. (B) RosA inhibits TCR-induced activation of Itk kinase activity. Jurkat cells were left unstimulated or stimulated with OKT3 cross-linking and then lysed. Total cell lysates were precipitated with anti-Itk mAb. Itk immunoprecipitates were divided into 2 parts, one for in vitro kinase assay (top panel) and the other for Western blotting (bottom panel). Itk kinase assay was performed as described in “Materials and methods.” Equivalent immunoprecipitation of Itk (bottom panel) was confirmed by Western blotting. Data are representative of 2 independent experiments.