Overexpression of GEM-Cbl-b suppresses tyrosine phosphorylation of FcϵRI and Syk. (A) Control cells and cells overexpressing GEM-Cbl-b were primed with anti-DNP IgE and then stimulated with 30 ng/mL antigen DNP-BSA for the indicated times. Cell lysates (3 × 106 cells) were solubilized in 1% Triton lysis buffer and immunoprecipitated with anti-FcϵRIβ mAb. The immunoprecipitates were separated by SDS-PAGE and analyzed by the immunoblotting with anti-pTyr mAb, anti-FcϵRIβ mAb. (B) Cells were solubilized in 1% Triton lysis buffer and immunoprecipitated with anti-Lyn antibody.Anti-Lyn immunoprecipitates were subjected to the in vitro protein kinase assay by using enolase as an external substrate. Radiolabeled proteins were separated by SDS-PAGE and visualized by autoradiography. Immunoprecipitated Lyn was analyzed by immunoblotting with anti-Lyn antibody. (C) Cells were solubilized in the denature buffer and immunoprecipitated with anti-pTyr mAb. The immunoprecipitates and detergent-soluble cell lysates were analyzed by the immunoblotting with anti-pTyr mAb and anti-Syk antibody. The numbers at the bottom of the figures are the normalized densitometric analysis of FcϵRIβ,FcϵRIγ, and Syk phosphorylation. Similar results were obtained when the other cloned lines were examined. The results were representative of 5 experiments.
