Defective Lyn/Btk/PLC-γ2 pathway in 4-1BB^-/- mast cells. (A) Wt and 4-1BB^-/- BMMCs that had been sensitized overnight with anti-DNP IgE were stimulated with 100 ng/mL DNP₂₃-HSA for the indicated periods. Cell lysates were directly analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies (A,C-D) or first immunoprecipitated with specific antibodies and immune complexes analyzed by SDS-PAGE and immunoblotting (B,E). In vitro kinase assays were also performed on immune complexes of Lyn and Fyn (C). (E) IgE-sensitized wt BMMCs were stimulated with 10 ng/mL DNP₂₃-HSA for 6 hours to induce 4-1BB expression, then restimulated with 100 ng/mL DNP₂₃-HSA for the indicated periods. Results shown are representative of at least 2 experiments.