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. 2013 Sep 9;191(7):3553–3562. doi: 10.4049/jimmunol.1300081

FIGURE 2.

FIGURE 2.

Cell surface binding of recombinant KIR3DL2-D1D2stem to HLA-F and MHC-I. (A) Soluble KIR3DL2-D1D2stem-bio binding to activated T cell clone 7D9 is blocked by anti–HLA-F and anti–MHC-I mAbs. FACS profile of refolded KIR3DL2-D1D2 staining activated 7D9 without (solid lines) and following addition (dashed lines) of anti–HLA-F mAb 3D11 (indicated within brackets). Control is clone 7D9 before activation stained with KIR3DL2-D1D1stem-bio construct (gray). To the right is a FACS showing the same clone stained with 3D11 (dashed lines) and HCA2 (solid lines), with control 16G1 in gray shading. T cells were stimulated with IL-2 and anti-CD3 for 4 d before staining. The graph immediately below shows a titration of KIR3DL2-D1D2-stem-bio staining of clone 7D9 before and after activation and with blocking by anti–MHC-I mAb HCA2 or anti–HLA-F mAb 3D11 (conditions indicated in the legend). (B) Western analysis with the indicated mAbs of gel fractionated after pull-down with KIR3DL2-D1D2stem-His. Five cell lines were incubated with KIR3DL2-D1D2stem-His and pull-downs performed followed by Western blot analysis with HCA2 and 3D11. In the panels below, cell lines were examined for surface expression of MHC-I HC (HCA2, dashed line), HLA-F (3D11, solid line), and MHC-I complex (W6/32, dotted line). Control mAb (QA-1) is in gray fill.