Figure 2.
HLA class II molecules transport DBY protein to the cell surface without fragmenting into peptides. (A) Flow cytometry of cell surface expression of H-Y antigens (DBY, EIF1AY, RPS4Y, UTY, and ZFY). Mock (shaded histogram), H-Y antigens (blue line), or H-Y antigens with HLA-DRB1∗15:02, HLA-DRB1∗09:01, or HLA-DRB1∗12:01 (red line) were transfected into 293T cells. All H-Y antigens were fused with FLAG-tag at the N-terminus and detected using the anti-FLAG-tag antibody. The results of cell surface analyses are shown. (B) Coimmunoprecipitation (Co-IP) assay of protein–protein interactions between full-length DBY and HLA-DRB1∗15:02. DBY was fused with FLAG-tag at the N-terminus and Myc-tag at the C-terminus. FLAG-DBY-Myc with HLA-DRB1∗15:02, FLAG-DBY-Myc, HLA-DRB1∗15:02, or mock was transfected into 293T cells. Transfected cells were lysed, and the Co-IP assay was performed using anti-HLA-DR antibody (left) and anti-FLAG-tag antibody (right). The resulting IP was blotted with the indicated antibodies. (C) PLAs to visualize protein–protein interactions between Myc-tagged DBY and HLA-DR. Colocalization of Myc-tagged DBY and HLA-DR induced PLA signals (red). Mock (upper left) or Myc-tagged DBY with HLA-DRB1∗15:02 (upper right), HLA-DRB1∗09:01(lower left), or HLA-DRB1∗12:01 (lower right) was transfected into 293T cells and analyzed. Nuclei were stained with DAPI. (D) Flow cytometry of cell surface DBY expression with or without Ii. Mock (shaded histogram), Ii with DBY and HLA class II (blue line), or DBY with HLA class II (red line) was transfected into 293T cells. Analyses of HLA-DRB1∗15:02 (left), HLA-DRB1∗09:01 (middle), and HLA-DRB1∗12:01 (right) are shown. Mock: pSI vector. Scale bars: 20 μm (C). Original magnification, ×400 (C). All data are representative of 3 independent experiments. DAPI, 4',6-diamidino-2-phenylindole.

HLA class II molecules transport DBY protein to the cell surface without fragmenting into peptides. (A) Flow cytometry of cell surface expression of H-Y antigens (DBY, EIF1AY, RPS4Y, UTY, and ZFY). Mock (shaded histogram), H-Y antigens (blue line), or H-Y antigens with HLA-DRB1∗15:02, HLA-DRB1∗09:01, or HLA-DRB1∗12:01 (red line) were transfected into 293T cells. All H-Y antigens were fused with FLAG-tag at the N-terminus and detected using the anti-FLAG-tag antibody. The results of cell surface analyses are shown. (B) Coimmunoprecipitation (Co-IP) assay of protein–protein interactions between full-length DBY and HLA-DRB1∗15:02. DBY was fused with FLAG-tag at the N-terminus and Myc-tag at the C-terminus. FLAG-DBY-Myc with HLA-DRB1∗15:02, FLAG-DBY-Myc, HLA-DRB1∗15:02, or mock was transfected into 293T cells. Transfected cells were lysed, and the Co-IP assay was performed using anti-HLA-DR antibody (left) and anti-FLAG-tag antibody (right). The resulting IP was blotted with the indicated antibodies. (C) PLAs to visualize protein–protein interactions between Myc-tagged DBY and HLA-DR. Colocalization of Myc-tagged DBY and HLA-DR induced PLA signals (red). Mock (upper left) or Myc-tagged DBY with HLA-DRB1∗15:02 (upper right), HLA-DRB1∗09:01(lower left), or HLA-DRB1∗12:01 (lower right) was transfected into 293T cells and analyzed. Nuclei were stained with DAPI. (D) Flow cytometry of cell surface DBY expression with or without Ii. Mock (shaded histogram), Ii with DBY and HLA class II (blue line), or DBY with HLA class II (red line) was transfected into 293T cells. Analyses of HLA-DRB1∗15:02 (left), HLA-DRB1∗09:01 (middle), and HLA-DRB1∗12:01 (right) are shown. Mock: pSI vector. Scale bars: 20 μm (C). Original magnification, ×400 (C). All data are representative of 3 independent experiments. DAPI, 4',6-diamidino-2-phenylindole.

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