Btk:ER activation is sufficient to induce ERK and JNK activation, and apoptosis, in Btk-deficient DT40 cells. A, Btk:ER-induced ERK MAPK activation. Cells were lysed as a function of the indicated times (minutes) after addition of 4-HT (1 μM), anti-μ mAb M4 (4 μg/ml), or phorbol 12-myristate 13-acetate (PMA, 100 ng/ml) as a positive control. ERK1 and ERK2 were immunoprecipitated with ERK1 and ERK2 antisera. Immune complex kinase assays were performed using myelin basic protein as an exogenous substrate (top panel). Samples were immunoblotted with anti-ERK1 and ERK2 antisera to show equal loading (bottom panel). ERK1 and ERK2 were not resolved as individual bands in these experiments, and ERK2 was the major species detected (data not shown). B, Btk:ER-induced JNK phosphorylation. Cells were lysed as a function of the indicated times (minutes) after addition of 4-HT (1 μM) and/or anti-μ mAb M4 (4 μg/ml), or PMA (100 ng/ml) and ionomycin (250 ng/ml) as a positive control. Whole cell lysates were immunoblotted with anti-active JNK phosphospecific antiserum. C, Btk:ER-induced apoptosis. Cells expressing either WT or kinase inactive Btk:ER were cultured for 24 h with 4-HT (1 μM) and/or anti-μ mAb M4 (2 μg/ml). Apoptosis was measured by TUNEL assay. The percentage of TUNEL-positive cells was calculated with reference to a negative control TUNEL reaction in the absence of terminal transferase. Negative controls yielded less than 1% TUNEL-positive cells in all cases. These data are representative of at least three independent stable clones.