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Physical association of Fc receptor γ chain homodimer with IgA receptor
Kan Saito, DMDa, b, Katsuhiro Suzuki, MSa, Hironori Matsuda, BSa, Ko Okumura, MD, PhDa, Chisei Ra, MD, PhDa Journal of Allergy and Clinical Immunology Volume 96, Issue 6, Pages (December 1995) DOI: /S (95) Copyright © 1995 Mosby, Inc. Terms and Conditions
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FIG. 1 FcR family. Fc receptors have the FcRγ chain as a common signal-transducing subunit, which contains a unique motif, ARAM. Only FcγRIIA possesses ARAM in its own cytoplasmic domain. (Y)-to-(Y), Cytoplasmic domain containing common signal-transducing motif, ARAM; Y, tyrosine residue. Journal of Allergy and Clinical Immunology , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions
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FIG. 2 ARAM, common motif in signal-transducing subunits of antigen recognition receptors. Antigen recognition receptors, TCR, BCR, and FcR, are composed of multiple subunits in which the signal-transducing function is mapped to the common motif DExxYxxLIxxxxxxxYxxLI (ARAM) in the cytoplasmic domain of each signal-transducing subunit. Journal of Allergy and Clinical Immunology , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions
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FIG. 3 Association of the FcαRα with a 20 kd protein. U937 cells were metabolically labeled with 35 S-cysteine for 24 hours in the presence of PMA and IFN-γ and solubilized with lysis buffer containing 1% digitonin. Cell lysates were immunoprecipitated with an anti-FcαRα mAb (A59), anti-FcRγ antibody, or control antibodies. Immunoprecipitates were subjected to SDS-PAGE (10% to 20% gradient gel) under unreduced conditions. Lane 1, Normal rabbit serum (NRS); lane 2, anti-FcRγ antibody (γ); lane 3, mlgG1 (G1); lane 4, anti-FcαRα mAb (A59). Journal of Allergy and Clinical Immunology , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions
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FIG. 4 Immunoblot analysis of FcRγ chain. U937 cell lysates were immunoprecipitated with an anti-FcαRα mAb (A59), anti-FcRγ antibody (γ), or control antibodies. The immunoprecipitates were resolved on SDS-PAGE (10% to 20% gradient gel) under unreduced (A) or reduced (B) conditions and transferred onto PVDF membranes for immunoblotting. The membranes were probed with anti-FcRγ antibody, and the protenin species bound to the anti-FcRγ antibody were detected with peroxidase-conjugated goat anti-rabbit IgG and an enhanced chemiluminescence kit. Lane 1 Normal rabbit serum (NRS); lane 2, anti-FcRγ antibody (γ); lane 3, mlgG1 (G1); lane 4, anti-FcαRα mAb (A59). Journal of Allergy and Clinical Immunology , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions
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FIG. 4 Immunoblot analysis of FcRγ chain. U937 cell lysates were immunoprecipitated with an anti-FcαRα mAb (A59), anti-FcRγ antibody (γ), or control antibodies. The immunoprecipitates were resolved on SDS-PAGE (10% to 20% gradient gel) under unreduced (A) or reduced (B) conditions and transferred onto PVDF membranes for immunoblotting. The membranes were probed with anti-FcRγ antibody, and the protenin species bound to the anti-FcRγ antibody were detected with peroxidase-conjugated goat anti-rabbit IgG and an enhanced chemiluminescence kit. Lane 1 Normal rabbit serum (NRS); lane 2, anti-FcRγ antibody (γ); lane 3, mlgG1 (G1); lane 4, anti-FcαRα mAb (A59). Journal of Allergy and Clinical Immunology , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions
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FIG. 5 Absorption of FcαRα with FcRγ. U937 cell lysates (metabolically labeled with 35 S-cysteine) were subjected to a serial immunoprecipitation with anti-FcRγ antibody(γ) and an anti-FcαRα mAb (A59). The immunoprecipitates were resolved on SDS-PAGE(10% to 20% gradient gel) under unreduced (A) or reduced (b) conditions. Lane 1, Normal rabbit serum (NRS); lane 2, anti-FcRγ antibody (γ); lane 3, mlgG1 (G1); lane 4, anti-FcαRα mAb (A59); lane 5, preabsorbed with anti-FcRγ antibody (A59*). Journal of Allergy and Clinical Immunology , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions
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FIG. 5 Absorption of FcαRα with FcRγ. U937 cell lysates (metabolically labeled with 35 S-cysteine) were subjected to a serial immunoprecipitation with anti-FcRγ antibody(γ) and an anti-FcαRα mAb (A59). The immunoprecipitates were resolved on SDS-PAGE(10% to 20% gradient gel) under unreduced (A) or reduced (b) conditions. Lane 1, Normal rabbit serum (NRS); lane 2, anti-FcRγ antibody (γ); lane 3, mlgG1 (G1); lane 4, anti-FcαRα mAb (A59); lane 5, preabsorbed with anti-FcRγ antibody (A59*). Journal of Allergy and Clinical Immunology , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions
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FIG. 6 Transient expression of human FcαR on transfectants. Human FcαRα and/or human FcRγ cDNAs were introduced into COS-7 cells. The transfectants were stained with an anti-FcαRα mAb (A59) and fluorescein isothiocyanate-labeled anti-mlgG1 mAb (second antibody) and analyzed by an immunofluorescence cell sorter for the expression of FcαR. A, Native COS-7 cells. B, FcαRα-introduced transfectants. C, FcRγ-introduced transfectants, D, FcαRα- and FcRγ-cointroduced transfectants. Solid line,A59; dotted line,second antibody only. Journal of Allergy and Clinical Immunology , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions
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FIG. 7 Physical association of FcRγ with FcαRα in stable stansfectants. FcαR-expressing stable transfectants were established by introducing human FcαRα and/or human FcRγ cDNA into CHO cells. Cell lysates from each transfectant were immunoprecipitated with an anti-FcαRα mAb (A59), anti-FcRγ antibody (γ), or control antibodies. Immunoprecipitates were resolved on SDS-PAGE (10% to 20% gradient gel) under unreduced (A) or reduced (B) conditions and then transferred onto PVDF membranes for immunoblotting probed with anti-FcRγ antibody as in Fig. 4. Lane 1, Normal rabbit serum (NRS); lane 2, anti-FcRγ antibody (γ); lane 3, mlgG1 (G1); lane 4, anti-FcαRα mAb (A59) CHO/FcαRα+FcRγ: CHO transfectants with FcαRα and FcRγ. CHO/FcαRα: CHO transfectants with FcαRα alone. Journal of Allergy and Clinical Immunology , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions
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FIG. 7 Physical association of FcRγ with FcαRα in stable stansfectants. FcαR-expressing stable transfectants were established by introducing human FcαRα and/or human FcRγ cDNA into CHO cells. Cell lysates from each transfectant were immunoprecipitated with an anti-FcαRα mAb (A59), anti-FcRγ antibody (γ), or control antibodies. Immunoprecipitates were resolved on SDS-PAGE (10% to 20% gradient gel) under unreduced (A) or reduced (B) conditions and then transferred onto PVDF membranes for immunoblotting probed with anti-FcRγ antibody as in Fig. 4. Lane 1, Normal rabbit serum (NRS); lane 2, anti-FcRγ antibody (γ); lane 3, mlgG1 (G1); lane 4, anti-FcαRα mAb (A59) CHO/FcαRα+FcRγ: CHO transfectants with FcαRα and FcRγ. CHO/FcαRα: CHO transfectants with FcαRα alone. Journal of Allergy and Clinical Immunology , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions
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