Hck deficiency results in reduced activities of positive regulatory molecules. IgE-sensitized WT and hck^−/− cells were stimulated with 100 ng/mL DNP₂₃-HSA for the indicated periods. (A) Syk was immunoprecipitated (indicated by thick vertical line on the right of gel) from cleared cell lysates and immune complexes subjected to in vitro kinase assays using GST-HS1 as a substrate. Portion of the autoradiogram including GST-HS1 phosphorylation is shown. Cell lysates were directly analyzed by SDS-PAGE and immunoblotting with anti-Syk or anti-phospho-Btk (Tyr223). The pBtk blot was reprobed with anti-Btk antibody. (B) Cell lysates were directly analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. The same blots were reprobed with antibodies that detect antigens irrespective of their phosphorylation status. (C) Immunoprecipitated JNK1 (indicated by thick vertical line on the right of gel) was subjected to in vitro kinase assays. Representative results from 2 experiments are shown.