Figure 7.

The UL16 transmembrane and cytoplasmic domains are involved in the intracellular retention of UL16, whereas the UL16 ectodomain is required for intracellular retention of ULBP2. (A) EL4 cells or EL4 cells transduced with a VSVG-pseudotyped retrovirus encoding UL16 and GFP were incubated in the presence or absence of 0.1% saponin to permeabilize cells. Cells were stained with anti-UL16 M230 or an isotype control antibody, fixed with 2% paraformaldehyde, and analyzed via flow cytometry. The histograms depict the mean fluorescence intensity (MFI) of cells stained with M230 minus the MFI of the cells stained with the control antibody. The results shown are representative of three separate experiments. (B) EL4 cells or EL4 cells transduced with a VSVG-pseudotyped retrovirus encoding the IL-4R/UL16 chimeric protein and GFP were stained as described above with anti-murine IL-4R M2 or an isotype control antibody. The histograms shown depict the MFI of cells stained with M2 minus the MFI of the cells stained with the isotype-matched control antibody. The results shown are representative of three separate experiments. (C) ULBP2⁺ EL4 cells were transduced with VSVG-pseudotyped retroviruses encoding UL16 and GFP, the UL16/IL-4R chimera and GFP, and the IL-4R/UL16 chimera and GFP. Transduced cells were enriched by fluorescence-activated cell sorting for cells expressing GFP, stained with anti-UL16 or with anti–IL-4R M2, and analyzed by flow cytometry. (D) ULBP2⁺ EL4 cells were transduced with VSVG-pseudotyped retroviruses encoding UL16 and GFP, the UL16/IL-4R chimera and GFP, and the IL-4R/UL16 chimera and GFP. Transduced cells were enriched by fluorescence-activated cell sorting of cells expressing GFP, stained with anti-ULBP2 M311, and analyzed by flow cytometry.