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. Author manuscript; available in PMC: 2013 Oct 1.
Published in final edited form as: J Immunol. 2013 Feb 25;190(7):3216–3224. doi: 10.4049/jimmunol.1202926

Figure 2. KIR3DL2Fc binds B272 more strongly than other HLA class I.

Figure 2

A. FACS staining of non-transfected LBL.721.221 (221) cells and 221 cells transfected with HLA-B27, HLA-A3 and HLA-B35 with KIR3DL2Fc. 221B27 cells stained with control DR5Fc fusion proteins is also shown. Geometric MFIs for staining of 221, 221B27, 221A3 and 221B35 were 18, 46, 22 and 17 respectively. Representative FACS stain from one of three independent experiments. B. Ratio of KIR3DL2Fc MFI:W632 MFI for staining of 221 transfectants. Ratios were calculated after subtracting the MFI for KIR3DL2Fc staining of parental 221 cells. Ratios were calculated from the mean MFIs from three independent experiments. C. Non-reducing SDS-PAGE gel and western blot with HC10 of precipitated proteins from 221B27 cells with control DR5Fc, lane I, and with KIR3DL2Fc from 221B27, lane II, and 221B35 transfected cells, lane III (left hand panel). Positions of multimeric, M, B27 dimer, B272, and monomeric B27 heavy chains, B27, are indicated. The right panel shows the same blot reprobed with HRP-conjugated anti-human Igs. The position of KIR3DL2Fc is indicated. Representative western blots from one of three independent experiments. D. Upper panel. Phosphotyrosine western blots of immunoprecipitates of KIR3DL2-transduced Jurkat T cells (lane I), or T cells stimulated with superantigen and parental 221 cells (lane II) or HLA-B35 (lane III), -A3 (lane IV), or -B27 transfectants (lane V). Lane VI: 221B27 cells alone. Lower panel. Western blot reprobed with anti-HA. Representative blots from one of three independent experiments.