The membrane recruitment of SLP-76 can occur independently of LAT1/LAT2 in NK cells. IL-2–expanded Ly49D-enriched WT or LAT/LAT2 DKO NK cells expressing GFP-tagged SLP-76, SLP-76.G2, SLP-76.RK, or SLP-76 G2.RK mutant were stimulated on an anti-Ly49D Ab-coated surface. (A) Clustering of SLP-76 at the plasma membrane was imaged by TIRF microscopy. (B) Summary of TIRF data represented as mean ± SEM of WT clustering in 3-8 independent experiments. Scale bar, 5 μm. (C) Ly49D⁺ splenic NK cells from WT or ADAP KO mice were enriched and expanded in IL-2 for 6 days. Expanded NK cells from WT or ADAP KO mice were stimulated with plate-immobilized Ct Ab or anti-Ly49D Ab for 4 hours in the presence of Monensin followed by detection of IFN and CD107a expression by flow cytometry. Ly49D expression of NK cells at the time of stimulation is shown. All plots are gated on CD4^–CD8^–NK1.1⁺ lymphocytes. (D) Data compiled from 3 Ly49D stimulation experiments were normalized to the WT NK cell response and expressed as mean ± SEM. (E) CFSE-labeled T cell-depleted WT or ADAP KO splenocytes were stimulated with plate-immobilized control Ab or anti-Ly49D Ab in the presence of 25 U/mL IL-2 for 3 days followed by flow cytometric analysis. Plots are gated on CD4^–CD8^–NK1.1⁺Ly49D⁺ NK cells. Cells to the left of gate have diluted CFSE and represent cells that have proliferated. One representative of 2 independent experiments is shown. (F) Expanded splenic NK cells were left unstimulated or stimulated with soluble anti-Ly49D Ab for 1 or 10 minutes and analyzed for total PLCγ2 (loading control), pAKT, and pERK1/2 by western blot. One representative of 2 independent experiments is shown. *P < .03 by t-test.