hOSCAR is associated with the ITAM-bearing adaptor FcRγ chain. (A) Stable HEK293T transfectants expressing an HA-tagged FcRγ were transfected with empty plasmid (mock) or plasmid-expressing CD16, hOSCAR wild-type (WT), or transmembrane mutant hOSCAR (R/H) and hOSCAR (R/V) substituting histidine and valine, respectively, for arginine. The HA-tagged FcRγ was detected by cell surface staining of the transfectants with anti-HA mAbs (shaded histograms) compared to in untransfected cells (open histograms). Dashed profiles indicate background staining with a control IgG1 mAb. (B) hOSCAR-transfected HEK293T with HA-tagged FcRγ (left panel) or FLAG-tagged DAP12 (center panel) and hOSCAR (R/V)-transfected HEK293T with HA-tagged FcRγ (right panel) were lysed in 1% digitonin buffer and immunoprecipitated with isotype-matched control mAbs (control IgG), anti-hOSCAR, or anti-tag mAbs. Western blot with biotin-conjugated anti-hOSCAR and ExtrAvidin-HRP allowed the detection of the receptor, while the presence of the adapters was revealed with HRP-conjugated anti-HA and anti-FLAG mAbs. HA-tagged FcRγ, but not FLAG-tagged DAP12, coimmunoprecipitates with hOSCAR. This association is lost with the R/V mutant. (C) Digitonin lysates from mono-DCs were incubated with isotype-matched control mAbs (control IgG), anti-hOSCAR, anti-CD54, anti-CD89 mAbs, or anti-FcRγ polyclonal Abs. The endogenous FcRγ in the immunoprecipitates was detected by Western blot with anti-FcRγ polyclonal Abs and HRP-conjugated donkey anti-rabbit Abs. The FcRγ coimmunoprecipitates with hOSCAR and CD89 in mono-DCs. The data are representative of 3 independent experiments.