NK cells acutely depleted of SLP-76 display a normal Ly49 receptor repertoire but still exhibit functional defects. (A) Splenic NK cells (CD3^–NK1.1⁺ lymphocytes) from control and SLP-76 cKO mice were analyzed for the expression of Ly49A, Ly49C/I, Ly49G2, Ly49D, and Ly49H and represented as mean percent positive ± SEM of 3 to 4 independent experiments. (B) Ly49D⁺ splenic NK cells from WT or SLP-76 cKO mice were enriched and expanded in IL-2 for 6 days. Expanded NK cells were stimulated by plate-bound isotype Ct Ab, anti-Ly49D Ab, or soluble PMA/ionomycin for 4 hours in the presence of Monensin followed by detection of IFN-γ and (C) CD107a expression by flow cytometry. Plots are gated on YFP⁺CD4^–CD8^–NK1.1⁺ lymphocytes. (D) Data compiled from 5 (IFN-γ) or 3 (CD107a) independent experiments were normalized to the control NK-cell response and expressed as mean ± SEM. * indicates statistical significance of P < .05 by paired t test. (E) The same NK cells wrtr stimulated with plate-bound anti-Ly49H or anti-NK1.1 antibody. IFN-γ and (F) CD107a surface expression was detected by flow cytometry. (G) The expanded NK cells were stimulated with soluble anti-Ly49D antibody for the indicated time and probed for pAKT and pERK1/2 by western blot analysis. PLCg2 served as the loading control. One representative of 2 independent experiments is shown.