Figure 2
iSEC1 and iSEC2 proteins exist as disulfide-linked homodimers and bind to CD200R but not CD200R-like receptors.
(a–c) NIH3T3 cells were infected with retroviral vectors encoding FLAG-tagged iSEC1 or iSEC2, or mock. In (a) transfectants were subjected to flow cytometric analysis for surface expression of iSEC1 or iSEC2 detected with anti-FLAG. Shaded histograms indicate control staining with isotype-matched control Ab. In (b) cell lysates of transfectants were subjected to SDS-PAGE under non-reducing (NR) or reducing (R) conditions, and then immunoblot analysis to detect FLAG-tagged iSEC1, as indicated by arrowheads. In (c) cell lysates of iSEC1 transfectants were treated with indicated units of N-glycosidase, and subjected to SDS-PAGE under reducing conditions, and then immunoblot analysis as in b. (d) NIH3T3 cells infected with retroviral vectors encoding FLAG-tagged CD200R family proteins, or mock were stained with anti-FLAG (open histograms) or isotype-matched control Ab (shaded histogram) to demonstrate surface expression of FLAG-tagged proteins (upper panels). In lower panels, the transfectants were incubated with iSEC1-IgFc fusion proteins (open histograms) or control mock IgFc (shaded histograms), followed by detection of bound fusion proteins with anti-IgG1. (e) NIH3T3 transfectants expressing mock control, CD200, iSEC1 or iSEC2 were incubated with CD200R-IgFc (open histograms) or control FLAG-IgFc (shaded histograms) fusion proteins, followed by detection of bound fusion proteins with anti-IgG1. (f) MC/9 transfectants expressing RFP and CD200R (Supplementary Fig. 4) were co-cultured for 24 h with 38B9 transfectants expressing GFP together with CD200, iSEC1, or iSEC2 (Supplementary Fig. 4), and the formation of red (RFP) and green (GFP) cell aggregates was examined under fluorescence microscope. Bar = 100 μm. Data shown in (a–f) are representative of at least three independent experiments.
