Induction of LAT phosphorylation by Ly49D expressed in human NK cells and RNKD2.38. Human NK cells were infected overnight with vaccinia carrying the Ly49D gene. (A) Vaccinia infected cells were stained for expression of CD56, Ly49D and CD16 and analyzed using flow cytometry. (B) The infected NK cells were divided into 5 samples and stimulated for 2 minutes at 37°C with the F(ab′)2 antibodies as indicated. Phosphorylation of immunoprecipitated LAT was determined by Western blot (pTyr, top panel) and the amount of LAT in each lane verified with anti-LAT (bottom panel). Controls were immunoprecipitated with preimmune sera = C. (C) RNKD2.38 cells (107) were stimulated with anti-Ly49D antibody (4E5), for 1, 5, or 10 minutes at 37°C. Phosphorylation and LAT expression were determined as in panel B. (D) FcϵRIγ was immunoprecipitated from RNKD2.38 cells stimulated with anti-Ly49D (4E5) or pervanadate (VO5). Proteins were resolved by SDS-PAGE and immunoblotted with antiphosphotyrosine. Results are representative of at least 2 independent experiments.
