Ly49Q was internalized with MHC class I, and its localization was regulated in an ITIM- and tyrosine phosphatase-dependent manner. (A) Colocalization of Ly49Q with H-2K^b in intracellular vesicular compartments. Peritoneal exudation macrophages were prepared from Ly49Q Tg mice and examined for Ly49Q (red) and MHC class I (green) by immunohistochemical analyses with a confocal microscope. FLAG-tagged Ly49Q in Tg mice was detected with an anti-FLAG antibody. (B) Binding of H-2K^b tetramer to pDCs. Cells enriched in bone marrow pDCs were obtained by AutoMACS using an anti–plasmacyloid DC Ag-1 antibody, and the binding of PE-conjugated H-2K^b tetramer was examined by flow cytometry. In the absence of β₂m, binding of the H-2K^b tetramer in trans was detectable due to loss of the cis interaction. The tetramer binding was abrogated by an anti-Ly49Q antibody. (C) Removal of β₂m from the cell surface by acid treatment. Bone marrow cells were treated with citrate buffer (0.133 M citric acid and 0.066 M Na₂HPO₄, pH 3.3) at 20°C for the indicated periods. Removal of β₂m from the cell surface was confirmed by a decreased fluorescence intensity of anti-β₂m antibody staining. CD11c⁺ cells were gated and analyzed. (D) Binding of H-2K^b tetramer before and after acid treatment. H-2K^b tetramer bound in trans to Ly49Q after the removal of β₂m from the cell surface, indicating that the cis interaction between Ly49Q and H-2K^b was still maintained, and the interaction was β₂m independent and acid resistant. (E) ITIM and tyrosine phosphatase dependence of Ly49Q redistribution. WEHI3 transfectants expressing Ly49Q-WT or Ly49Q-YF were incubated at 37°C in the presence or absence of the indicated inhibitors of membrane trafficking. The cells were then fixed and stained with an anti-FLAG antibody to visualize Ly49Q.