Expression of Btk:ER, a conditional form of Btk, in Btk-deficient DT40 cells. A, Diagrammatic representation of Btk:ER constructs. WT and kinase inactive (K430E) forms of Btk were fused at their COOH-terminus to the hormone-binding domain of the estrogen receptor (ER). B, Immune-complex kinase assay and Western blot analyses of Btk:ER constructs. Btk-deficient DT40 cells were stably transfected with WT Btk:ER or K430E Btk:ER and clones selected that expressed equal levels of Btk:ER. Cells were lysed and Btk:ER was immunoprecipitated with specific Btk antiserum. Immune complex kinase assays were performed using GST-Igα as an exogenous substrate (top panel), in addition to immunoblotting with antisera specific for Btk (middle panel) and ER (lower panel). C, Activation of Btk:ER in vivo by 4-HT. Cells expressing either WT or kinase inactive Btk:ER were stimulated with vehicle control, 4-HT (1 μM for 30-min) or anti-μmAb M4 (4 μg/ml for 1-min). Cells were lysed and Btk:ER was immunoprecipitated with specific Btk antiserum. Whole cell lysates were blotted with mAb 4G10 (top panel). Btk mmunoprecipitates were blotted with mAb 4G10, phosphospecific Btk Y223 antiserum, or Btk antiserum (lower panels).