Figure 3.

BCR-induced activation of Syk and Btk. (A) Tyrosine phosphorylation profiles of wild-type and Cbl-b–deficient (C3-3) DT40 cells. Whole cell lysates were prepared at the indicated time points after stimulation of 4 μg/ml M4. The lysates were subjected to Western blot analysis with anti-phosphotyrosine mAb. (B) Tyrosine phosphorylation and kinase activity of Syk. Wild-type or Cbl-b–deficient (C3-3) DT40 cells were lysed at the indicated time points after stimulation of 4 μg/ml M4. Anti-Syk immunoprecipitates from the lysates were equally divided, and aliquots (one third of the lysates) were subjected to in vitro kinase assay with GST-Igα as a substrate (top), and the remaining immunoprecipitates were analyzed by Western blot analysis with anti-phosphotyrosine mAb (middle) and anti-Syk Ab (bottom). (C) Kinase activity of Btk. Wild-type or Cbl-b–deficient DT40 cells expressing T7-tagged Btk were lysed at the indicated time points after stimulation of 4 μg/ml M4. Anti-T7 immunoprecipitates from the lysates were equally divided, and half of them were subjected to in vitro kinase assay with enolase as a substrate (top), and the remaining half were analyzed by Western blot analysis with anti-T7 mAb (bottom). (D) Tyrosine phosphorylation of Btk. Wild-type or Cbl-b–deficient DT40 cells expressing T7-tagged Btk were lysed in 0.2% NP-40 lysis buffer at the indicated time points after stimulation of 4 μg/ml M4. Anti-T7 immunoprecipitates from the lysates were used for Western blot analysis with anti-phosphotyrosine mAb (top) and anti-T7 mAb (bottom). (E) Tyrosine phosphorylation of BLNK. Wild-type or Cbl-b–deficient (C3-3) DT40 cells (5 × 10⁶) were lysed in 1% NP-40 lysis buffer at the indicated time points after stimulation of 4 μg/ml M4. Anti-BLNK immunoprecipitates from the lysates were subjected to Western blot analysis with anti-phosphotyrosine mAb (top) and anti-BLNK Ab (bottom). All experiments were performed more than three times.