FIG. 3.

Effects of wortmannin on Akt phosphorylation and membrane distribution and kinase activity of Itk. JTAg T cells were treated with wortmannin (100 nM) for the indicated times prior to stimulation by cross-linking CD3 (OKT3 ascites). (A) Whole-cell lysates were immunoblotted with an anti-phospho-Akt antibody which recognizes the Ser-473-phosphorylated, active form of Akt. (B) The cytosolic (c) and membrane (m) fractions were prepared from the same samples and immunoblotted with an anti-Itk monoclonal antibody (2F12). The percentage of total Itk localized to the membrane fraction was determined by densitometric analysis of the X-ray films. (C) Itk was immunoprecipitated with rabbit polyclonal anti-Itk antisera from 10⁷ cells treated with or without wortmannin (100 nM) and OKT3 as indicated and was subjected to an in vitro kinase assay. The inset shows an Itk blot of the Itk immunoprecipitate, indicating that equal amounts of Itk were used in the kinase assay.