LAT and NTAL are expressed in mouse NK cells but are dispensable for splenic NK cell maturation. (A) Lat and Ntal expression levels are expressed as relative units of indicated mRNA normalized using HPRT transcripts. Samples included purified CD4⁺ T cells (T), sorted (CD3^–CD4^–CD8^–DX5⁺) NK cells isolated from unstimulated (NK resting) and polyIC stimulated (NK polyIC) mice, sorted (CD3^–NK1.1⁺) LAK, enriched B220⁺ B cells (B), all derived from C57BL/6 mice, or total splenocytes isolated from double LAT/NTAL^KO mice. Data (mean ± SD) are representative of 3 independent experiments. (B) LAT and NTAL expression was analyzed on C57BL/6 (CTR) and LAT/NTAL^KO spleen lymphocytes by intracellular staining gating on T cells (CD3⁺NK1.1^–), NK cells (CD3^–NK1.1⁺), and B cells (CD3^–NK1.1^–). Staining performed with isotype control mAb (dotted line) has been overlaid to staining obtained using specific anti-LAT or anti-NTAL mAb (bold line). Data shown are representative of 3 independent experiments. (C) LAT and NTAL expression was analyzed by immunoblotting on lysates prepared from (1) 5 × 10⁶ total C57BL/6 splenocytes, (2) 5 × 10⁶ total splenocytes derived from LAT^KO mice, (3) 5 × 10⁶ total splenocytes derived from NTAL^KO mice, (4) wild-type thymocytes (1 × 10⁶), (5) purified (CD3^–CD4^–CD8^–DX5⁺) NK cells (1 × 10⁶), and (6) enriched (B220⁺) B cells (1 × 10⁶). (D) The numbers of splenic NK cells isolated from indicated mice are represented. Dots indicate individual mice. Horizontal bars indicate mean values. Statistical analysis was performed using unpaired Student t test. (E) The cell-surface expression of indicated NK cell markers was analyzed on CD3^–MHC class II^–DX5⁺ splenocytes isolated from represented mice. Indicated mice also express comparable levels of Ly49A marker (data not shown). Data are representative of 4 independent experiments.