LIME associates with Lyn, Grb2, and PLC-γ2. (A) Expression plasmids encoding LIME-flag were transfected into A20 cells. Subsequently, cells were activated by cross-linking with F(ab′)2 goat anti-mouse IgG for the indicated times. Cell lysates were subjected to immunoprecipitation using an antiflag Ab, and the precipitates were analyzed by Western blotting using anti-Lyn, anti-Grb2, anti-PLC-γ2, antiphosphotyrosine (4G10), antiflotillin-2, and antiflag Abs. In antiphosphotyrosine blot (fifth panel), a 32-kDa band corresponding to the size of LIME was detected upon BCR stimulation. (B) Mapping of the binding sites for association with Lyn. His-tagged Lyn was coexpressed with flag-tagged tyrosine mutants of LIME (tyrosine→phenylalanine) in 293T cells. Lysates were analyzed by immunoprecipitation using an antiflag Ab followed by immunoblotting with an anti-His, antiflag, or antiphosphotyrosine (4G10) Ab. (C) Mapping of the binding sites for association with Grb2. Flag-tagged tyrosine mutants of LIME were coexpressed with Grb2 and Lyn in 293T cells. Cell lysates were processed as described in panel B. (D) Mapping of the binding sites for association with PLC-γ2. Flag-tagged tyrosine mutants of LIME were coexpressed with PLC-γ2 and Lyn in 293T cells. Cell lysates were processed as described in panel B.
