Btk:ER activation is sufficient to induce PLCγ2 phosphorylation and calcium mobilization in Btk-deficient DT40 cells. A, Btk:ER-induced PLCγ2 phosphorylation. Cells expressing either WT or kinase inactive Btk:ER were lysed as a function of the indicated times (minutes) after addition of 4-HT (1 μM) or BCR cross-linking with anti-μ mAb M4 (4 μg/ml). PLCγ2 was immunoprecipitated with anti-PLCγ2 antiserum and immunoblotted with anti-phosphotyrosine mAb (APT, top panel) and with anti-PLCγ2 antiserum (bottom panel). B, BCR-induced PLCγ2 phosphorylation in Btk-deficient versus Btk-sufficient cells. The experiment was performed as described for Fig. 2A. Lanes 1-8 show anti-μ-induced PLCγ2 phosphorylation in Btk-deficient DT40 cells and in Btk-deficent cells expressing mouse Btk, which were described previously . Lanes 9-12 show 4-HT-induced PLCγ2 phosphorylation in Btk-deficient cells expressing Btk or Btk:ER. Lanes 13-15 show a Btk blot of whole cell lysates for Btk-deficient cells, Btk-deficient cells expressing Btk, and Btk-deficient cells expressing Btk:ER. C, Btk:ER-induced calcium mobilization. Intracellular free calcium levels in indo-1-loaded cells were monitored over an 8-min period by FACS. 4-HT (1 μM) and/or anti-μ mAb M4 (2 μg/ml) were added at the 60 s time point. Relative intracellular calcium levels are shown on the y-axis. D, Sustained calcium mobilization is dependent on calcium influx. Intracellular calcium levels were monitored as described in panel B, in the presence or absence of 3 mM EGTA which chelates extracellular calcium. These data are representative of three independent stable clones.