DAP10 expression levels regulate the amount of NKG2D surface expression. Cells (293T) were transfected with either NKG2D (1.0 μg), DAP10 (1.0 μg), or 1.0 μg NKG2D and an increasing amount of DAP10 (0.3, 1.0 μg) cDNA. Forty-eight hours after transfection, the cells were analyzed for mRNA and protein levels of NKG2D and DAP10. (A) The NKG2D (left panel) and DAP10 (right panel) transcripts were analyzed by qRT-PCR with specific primer sets and normalized to actin RNA, and are presented in arbitrary units. Data shown are from 2 separate experiments. (B) The expression of NKG2D on the cell surface of 293T cells after transfection was analyzed by flow cytometry. The value of median fluorescence intensity of NKG2D is indicated in parentheses in each graph. Gray filled histograms represent isotype controls. Data are representative of 2 separate experiments. (C) NKG2D and DAP10 protein levels from total cell lysates of 293T transfectants were analyzed by Western blotting. Anti-actin immunoblotting was used as a loading control. The result shown is representative of 2 independent experiments.