Ligation of BCR induces tyrosine phosphorylation and activation of Tec. (A) Ramos cells were incubated with anti-IgM antiserum or control serum for 5 minutes. Proteins immunoprecipitated with anti-Tec rabbit antibody or normal rabbit serum (NRS) 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 kD) are indicated. The position of Tec is indicated by an arrow. A tyrosine phosphorylated protein of approximately 62 kD, coprecipitated with Tec, is also noticeable. The band above Tec (approximately 74 kD) is likely due to nonspecific reactivity, as it was also detected after BCR ligation in immunoprecipitates obtained with NRS. (B) Kinetics of BCR-mediated tyrosine phosphorylation of Tec. Ramos cells were incubated with anti-IgM antiserum for the times indicated (minutes). Tyrosine phosphorylation of Tec was assessed above. (C) BCR ligation increases Tec kinase activity. Ramos cells were stimulated as above for 5 minutes and Tec kinase activity was assessed by in vitro kinase assay. The position of Tec is indicated by the arrow.