FIG 2.
NCR1 is activated by reovirus. (A) FACS staining of Vero cells incubated for 14 h in the presence or absence of reovirus. The filled gray histogram depicts the background staining of Vero cells with the secondary MAb in the absence of reovirus. The background staining of Vero cells in the presence of reovirus was similar and is not shown. The background used here is identical to the background used in Fig. 1B, as the experiments were performed at the same time. The empty black histogram depicts the staining of Vero cells with NCR1-Ig. The empty gray histogram depicts the staining of Vero cells preincubated with reovirus and stained with NCR1-Ig. Shown are the results of one representative experiment out of three performed. (B) The median fluorescence intensity (MFI) of NCR1-Ig staining of uninfected and infected cells obtained from three different experiments. Each error bar represents the standard deviation (SD). Statistically significant differences are indicated. *, P < 0.05. (C) Coomassie staining of the NCR-1-Ig used in in panel A after gel electrophoresis under nonreducing conditions. The image was cropped and the background was adjusted for better clarity. (D) FACS staining of BW cells expressing NCR1-zeta (BW NCR1). The empty black histogram depicts staining with the anti-NCR1 MAb. The filled gray histogram depicts staining with the secondary MAb only. (E) BW cells or BW cells expressing NCR1-zeta (BW NCR1) were cocultured for 48 h with Vero cells preincubated for 14 h in the presence (reovirus) or absence (uninfected) of reovirus. IL-2 secretion was determined 24 h later by ELISA. Relative IL-2 secretion, determined as described in Materials and Methods, is shown. The mean values and SD of three independent experiments are shown. Statistically significant differences are indicated. *, P < 0.05. (F) Vero cells incubated with reovirus for 14 h were tested in a killing assay against mouse Ncr1+/gfp (Ncr1 Het) and Ncr1gfp/gfp (Ncr1 KO) NK cells. The effector-to-target cell ratio was 1:1. The mean values and SD of three independent experiments are shown. Statistically significant differences are indicated. *, P < 0.05; **, P < 0.01. In all flow cytometry experiments, antibodies and fusion proteins were incubated with target cells on ice. Fusion proteins were incubated for 2 h, and antibodies were incubated for 1 h.