FIG. 7.
Targeting of Itk to the membrane and sensitivity of Itk to CD3 stimulation requires an intact PH domain. Six micrograms of each pSRα-Itk-myc construct or vector was electroporated into 1.2 × 107 JTAg T cells to get about two- to threefold overexpression of the recombinant Itk over endogenous Itk. (A) The levels of Itk expression were monitored by Western blot analysis of the whole-cell lysates with an anti-myc antibody (9E10) (top) or an anti-Itk antibody (2F12) (bottom). (B) The cytosol (c) and membrane (m) fractions from pSR-Itk(WT) and pSR-Itk(R29C) transfectants were electrophoresed on a 6% Tris-glycine gel to resolve the endogenous and transfected Itk's and then immunoblotted with an anti-Itk antibody (2F12). (C) The recombinant Itk was immunoprecipitated with an anti-myc antibody (9E10) from 2 × 107 JTAg T cells transfected with either pSR-Itk(WT) or pSR-Itk(R29C) and was analyzed in an in vitro kinase assay.