Upon BCR cross-linking, LIME is phosphorylated by Lyn tyrosine kinase. (A) A20 cells were transfected with constructs encoding LIME-flag. Subsequently, cells were pretreated with either DMSO, PP1 (a Src family tyrosine kinase inhibitor), or piceatannol (a Syk inhibitor) and stimulated with F(ab′)2 goat anti-mouse IgG for 1 minute. LIME was immunoprecipitated from cell lysates using an antiflag Ab, and the precipitates were analyzed by immunoblotting with an antiphosphotyrosine (4G10) or antiflag Ab. As a control, the same cell lysates were analyzed by immunoblotting with an antiphosphotyrosine Ab (third panel). (B) LIME-flag was coexpressed with Lyn or Syk in 293T cells. Lysates were subjected to immunoprecipitation with an antiflag Ab, and the precipitates were analyzed by Western blotting with antiphosphotyrosine (4G10) and antiflag Abs.
