Figure 3.
Overexpression of Sab in DT40 cells interferes with BCR-induced, Btk-related signaling events. (A) BCR-induced tyrosine phosphorylation of whole-cell proteins in the mock and the Sab-overexpressed (+Sab) DT40 cells. After DT40 cells were stimulated with M4 for the indicated time periods, the cell lysates were immunoblotted with the anti-phosphotyrosine antibody 4G10. (B) Tyrosine phosphorylation of Btk after BCR engagement was reduced in Sab-overexpressed DT40 cells. DT40 cells (mock or Sab-overexpressed) were lysed before and after stimulation with M4 for 3 min. Btk in the lysates then was immunoprecipitated with the anti-Btk antibody 43-3B and immunoblotted with the anti-phosphotyrosine antibody 4G10. (Lower) Confirmation of equal amounts of the Btk protein in immunoprecipitates. (C) Calcium mobilization after BCR engagement. Intracellular free calcium levels in Fura-2 AM-loaded cells were monitored with a spectrophotometer after stimulation with M4 of the mock, Sab, or Sab(Δ30)-overexpressed and Btk-deficient DT40 cells. (D) Ins(1,4,5)P3 generations were detected by means of extracting soluble inositol from DT40 cells stimulated with M4 for the indicated time periods.