Most, if not all, mAbs against KIR2DL1 compete with each other for binding and compete with HLA class I ligands. Thus, in experiments with YTS-KIR2DL1 cells, we made use of a FLAG tag at the N terminus of KIR2DL1 so that the receptor could be ligated with an anti-FLAG mAb and stained with a noncompeting mAb. Anti-FLAG (clone 9A3, mouse IgG1, 1:40 dilution; Cell Signaling) and EB6–Alexa Fluor 647 were used to stimulate and stain these cells. Antibodies against the signaling proteins CrkII (3G11C1, mouse IgG2b, 1:300 dilution; Abcam), SHP-1 (255402, rat IgG2a, 10 μg/ml; R&D Systems), SHP-1 pY536 (rabbit polyclonal, 1:200 dilution; Acris Antibodies Gmbh), CrkL pY207, and CrkII pY221 (both rabbit polyclonal antibodies, 1:100 dilution; New England Biolabs) were detected using the appropriate cross-adsorbed secondary antibody (Alexa Fluor 568, 10 μg/ml for microscopy; Thermo Fisher Scientific). The directly conjugated anti-pCrkL-Y207 Alexa Fluor 488 antibody (clone K30-391.50.80, mouse IgG2a, 1:10 dilution; BD Biosciences) was used in confocal experiments. DX9, an anti-KIR3DL1 antibody (5 μg/ml; R&D Systems), and HP3E4, an anti-KIR2DL1 antibody (mouse IgM, 2.5 μg/ml; BD Pharmingen), were used to block cytotoxicity. Anti-CD28 (CD28.2) and anti–LFA-1 (HI111; both mouse IgG1, 5 μg/ml; eBioscience) were used to activate YTS cells for imaging. In other experiments, anti-NKp30/NCR3 (210847, mouse IgG2a, 5 μg/ml; R&D Systems) and anti–LFA-1 (HI111; mouse IgG1, 1.25 μg/ml; eBioscience) or ICAM-1 (1.25 μg/ml; produced in-house) was used to activate YTS and pNK cells for imaging and enzyme-linked immunosorbent assay (ELISA). HLA-C*0401 and HLA-B*5701 class I biotinylated monomers (National Institutes of Health Tetramer Core Facility) were coated onto slides at 5 and 40 μg/ml, respectively. KIR proteins interacting with HLA were detected using anti-FLAG Alexa Fluor 647 (clone L5, rat IgG2a, 10 μg/ml; BioLegend) for KIR2DL1 or anti-KIR3DL1 (177407, mouse IgG2a, 10 μl per test; R&D Systems) conjugated to Alexa Fluor 647. Staining of KIR on these surfaces was compared to the staining of KIR on slides coated at the same density with control proteins [bovine serum albumin (BSA) or mouse IgG2a] that did not ligate the receptor.

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