Figure 4
Figure 4. HRG does not directly interact with FcγR on THP-1 cells. (A) Effect of precoating THP-1 cells with a human FcγRI-specific blocking F(ab′)2 mAb (10.1, 40 μg/mL), a human FcγRIIA-specific blocking F(ab′)2 mAb (8.26, 40 μg/mL), or a human IgG1κ myeloma (200 μg/mL) on biotinylated HRGPID (100 μg/mL) binding to THP-1 cells. The effect of heparin (12.5 kDa, 50 μg/mL) on biotinylated HRGPID binding to THP-1 cells was also examined. Biotinylated HRGPID binding was detected by flow cytometry using PE-conjugated streptavidin. Representative flow cytometric histograms are shown, with filled histograms representing the PE-conjugated streptavidin only control and open blue and red histograms representing, respectively, biotinylated HRGPID binding in the absence or presence of the indicated treatments. Effect of precoating THP-1 cells with (B) HRGP or (C) HRGPID on the binding of biotinylated human IgG1κ myeloma to THP-1 cells. Biotinylated IgG binding was determined by flow cytometry using PE-conjugated streptavidin, with data being expressed as fold binding above background MFI. (D) Effect of precoating THP-1 cells with a human FcγRI-specific blocking F(ab′)2 mAb (10.1, 40 μg/mL) or a human FcγRIIA-specific blocking F(ab′)2 mAb (8.26, 40 μg/mL) on the binding of the copurified IgG in HRGP (100 μg/mL) to THP-1 cells. (E) Cell-surface expression of human FcγRIIA by the GAG-deficient CHO cell line (pgsA-745) stably transfected with human FcγRIIA containing either an arginine residue or a histidine residue at amino acid position 131 (FcγRIIA-arg131 and FcγRIIA-his131, respectively), with untransfected pgsA-745 cells being included as a negative control. Human FcγRIIA expression was detected by flow cytometry using a mouse F(ab′)2 anti–human FcγRIIA mAb (8.26) and a PE-conjugated sheep F(ab′)2 anti–mouse Ig Ab. Representative flow cytometric histograms are shown, with filled histograms representing a secondary Ab-only control and open black histograms representing cell-surface expression of human FcγRIIA. (F) Binding of the copurified IgG in HRGP (100 μg/mL) to pgsA-745 cells stably transfected with either human FcγRIIA-arg131 or FcγRIIA-his131, with untransfected pgsA-745 cells being included as a negative control. IgG binding in panels D and F was detected by flow cytometry. Data in panels D and F are expressed as fold binding above background MFI, with error bars representing SEM (n = 3). NS indicates not significant. ***P < .001.

HRG does not directly interact with FcγR on THP-1 cells. (A) Effect of precoating THP-1 cells with a human FcγRI-specific blocking F(ab′)2 mAb (10.1, 40 μg/mL), a human FcγRIIA-specific blocking F(ab′)2 mAb (8.26, 40 μg/mL), or a human IgG1κ myeloma (200 μg/mL) on biotinylated HRGPID (100 μg/mL) binding to THP-1 cells. The effect of heparin (12.5 kDa, 50 μg/mL) on biotinylated HRGPID binding to THP-1 cells was also examined. Biotinylated HRGPID binding was detected by flow cytometry using PE-conjugated streptavidin. Representative flow cytometric histograms are shown, with filled histograms representing the PE-conjugated streptavidin only control and open blue and red histograms representing, respectively, biotinylated HRGPID binding in the absence or presence of the indicated treatments. Effect of precoating THP-1 cells with (B) HRGP or (C) HRGPID on the binding of biotinylated human IgG1κ myeloma to THP-1 cells. Biotinylated IgG binding was determined by flow cytometry using PE-conjugated streptavidin, with data being expressed as fold binding above background MFI. (D) Effect of precoating THP-1 cells with a human FcγRI-specific blocking F(ab′)2 mAb (10.1, 40 μg/mL) or a human FcγRIIA-specific blocking F(ab′)2 mAb (8.26, 40 μg/mL) on the binding of the copurified IgG in HRGP (100 μg/mL) to THP-1 cells. (E) Cell-surface expression of human FcγRIIA by the GAG-deficient CHO cell line (pgsA-745) stably transfected with human FcγRIIA containing either an arginine residue or a histidine residue at amino acid position 131 (FcγRIIA-arg131 and FcγRIIA-his131, respectively), with untransfected pgsA-745 cells being included as a negative control. Human FcγRIIA expression was detected by flow cytometry using a mouse F(ab′)2 anti–human FcγRIIA mAb (8.26) and a PE-conjugated sheep F(ab′)2 anti–mouse Ig Ab. Representative flow cytometric histograms are shown, with filled histograms representing a secondary Ab-only control and open black histograms representing cell-surface expression of human FcγRIIA. (F) Binding of the copurified IgG in HRGP (100 μg/mL) to pgsA-745 cells stably transfected with either human FcγRIIA-arg131 or FcγRIIA-his131, with untransfected pgsA-745 cells being included as a negative control. IgG binding in panels D and F was detected by flow cytometry. Data in panels D and F are expressed as fold binding above background MFI, with error bars representing SEM (n = 3). NS indicates not significant. ***P < .001.

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