Ligation of CD19 in immature B cells induces tyrosine phosphorylation and activation of Btk. (A) Immature B-cell lines, RS4;11, 380, and NALM6 were incubated with anti-CD19 monoclonal antibody or isotype-matched nonreactive Ig for 15 minutes at 4°C followed by goat antimouse Ig antiserum for 1 minute at 37°C. Proteins immunoprecipitated with anti-Btk 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-Btk goat antibody (lower panel). The position of Btk is indicated. In RS4;11 and 380 cells, cross-linking of CD19 induced tyrosine phosphorylation of Btk, as 5-minute exposure to anti-IgM antibody did in Ramos cells.33-35 In NALM6 cells, Btk appeared to be strongly tyrosine phosphorylated before CD19 ligation and no increase in phosphorylation was detected. (B) Kinetics of CD19-mediated tyrosine phosphorylation of Btk. CD19 was cross-linked on RS4;11 cells for the times indicated (minutes). Tyrosine phosphorylation of Btk was assessed as above. (C) CD19 ligation increases Btk kinase activity. The 380 cells were stimulated as above for 1 minute and Btk kinase activity was assessed by in vitro kinase assay. Molecular mass markers (in kilodaltons) are indicated. The position of Btk is indicated by the arrow.