FIG. 5.

Antigen-induced tyrosine phosphorylation of FcɛRI, Syk, and LAT in cells expressing palmitoylation site-mutated Lyn. BMMC-Lyn^−/− were transfected with LynB-WT, LynB-CA, LynB-GCA, or GFP alone. The transfected and IgE-sensitized cells were activated or not with TNP-BSA for 5 min. The cells were lysed, and FcɛRI complexes, Syk, or LAT was immunoprecipitated from the postnuclear supernatants using Abs specific for IgE (A), Syk (B), or LAT (C). Proteins were resolved by SDS-PAGE and analyzed by immunoblotting (IB) with antiphosphotyrosine MAb PY-20-HRP. After stripping, MAbs specific for FcɛRI β subunit, Syk, and LAT were used to estimate the relative amounts of the immunoprecipitated (IP) protein. Arrows indicate the positions of the phosphorylated β and γ subunits of the FcɛRI (A), Syk (B), and LAT (C). (A) Positions of size markers are shown on the right (in kilodaltons). (D) Quantitative analysis of tyrosine phosphorylation of immunoprecipitated FcɛRI, Syk, and LAT from cells transfected with LynB-WT (WT), LynB-CA (CA), LynB-GCA (GCA), or GFP alone. Tyrosine phosphorylation of FcɛRI, Syk and LAT from cells transfected with LynB-WT was taken as 100%. Means ± SD were calculated from four independent experiments in each group. Data were normalized for different transfections efficiencies as determined by flow cytofluorometry.