Stimulation with a low concentration of antigen does not induce phosphorylation of FcϵRIβ or increase SHIP phosphorylation in hck^−/− cells despite increased Lyn activity. IgE-sensitized WT and hck^−/− cells were stimulated with 1 ng/mL DNP₂₃-HSA for the indicated periods. Cell lysates were directly analyzed by SDS-PAGE and immunoblotting with the indicated phospho-specific antibodies. The same blots were reprobed with antibodies that detect antigens irrespective of their phosphorylation status. (A, third and fourth rows) Immunoprecipitated Lyn was subjected to autophosphorylation assays. Comparable immunoprecipitations were confirmed by immunoblotting. (A, middle) Immunoprecipitated FcϵRIβ was analyzed by immunoblotting with anti-phosphotyrosine mAb and then reprobed with anti-FcϵRIβ mAb. Immunoprecipitations are indicated by thick vertical lines on the right of gels. Representative results from 2 experiments are shown.