Ligation of CD38 in RS4;11 cells induces tyrosine phosphorylation and activation of Tec, but not of Btk. (A) RS4;11 cells were incubated with anti-CD38 monoclonal antibodies, T16 and THB7, and isotype-matched nonreactive Ig for 5 minutes. Proteins immunoprecipitated with anti-Tec rabbit antibody were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was probed with antiphosphotyrosine antibody (pTyr; upper panel), then stripped and reprobed with anti-Tec rabbit antibody (lower panel). Molecular mass markers (in kilodaltons) are indicated. The position of Tec is indicated by an arrow. (B) CD38 ligation increases Tec kinase activity. RS4;11 cells were stimulated with THB7 for 5 minutes and Tec kinase activity was assessed by in vitro kinase assay. (C) RS4;11 cells were stimulated with anti-CD38 monoclonal antibodies as in (A). Tyrosine phosphorylation of Btk was examined as above. The position of Btk is indicated by an arrow. The presence of Btk in the immunoprecipitates was confirmed by stripping the blot and reprobing with anti-Btk goat antibody (lower panel).