isotype Search Results


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  • 85
    Millipore isotype control mabs
    Cell Surface Shed and Secreted, Soluble HLA-G Is Endocytosed into KIR2DL4-Containing Vesicles (A) Endocytosis of soluble HLA-G in resting NK cells. The 221 cells and 221 cells transfected with HLA-Cw3 (221-Cw3) were fixed, permeabilized, and stained with <t>mAb</t> F4/326. Resting NK cells were incubated at 37 °C for 120 min with soluble, refolded HLA-C or HLA-G. Cells were then fixed, permeabilized, and stained with reagents to detect HLA-C (F4/326) or HLA-G (G233) as indicated. (B) The NK cell line YTS-2DL4-gfp was loaded at 37 °C for 120 min with refolded HLA-G. Cells were fixed, permeabilized, and stained with mAb G233 to detect co-localization of soluble HLA-G with gfp-tagged KIR2DL4. (C) Recombinant soluble molecules of HLA-G but not HLA-C are endocytosed into 293T-2DL4-gfp cells. Refolded HLA-G and HLA-C were incubated with 293T-2DL4-gfp cells for 2 h. Cells were then fixed, permeabilized, and stained with either mAb G233 (to detect endocytosed HLA-G; upper) or mAb F4/326 (to detect endocytosed HLA-C; middle). (D) The 293T-2DL4-gfp cells were co-cultured with an equal number of 221 cells, 221 cells expressing transmembrane HLA-G (221-G), and 221 cells expressing a soluble isoform of HLA-G (221-sG) for 48 h. Adherent 293T-2DL4-gfp cells were fixed, permeabilized, and stained with mAb G233 followed by Alexa-568–conjugated secondary antibodies prior to acquisition of confocal images. Two 221-G cells are visible in the middle panel.
    Isotype Control Mabs, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher igg1 isotype control antibodies
    Inflammatory cell infiltrates in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA <t>IgG1</t> antibody. (A) Legend. (B,C) Bar graphs show mean ± SEM and individual data for inflammatory cell infiltrates (scores, U) accumulating (B) perivascularly / peribronchially or in the (C) alveoli / interstitium of the lungs. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p
    Igg1 Isotype Control Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/igg1 isotype control antibodies/product/Thermo Fisher
    Average 78 stars, based on 2 article reviews
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    igg1 isotype control antibodies - by Bioz Stars, 2019-05
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    99
    BioLegend isotype igg
    Serum C-peptide levels of the mice at 48 h after <t>anti-CD3</t> or isotype <t>IgG</t> treatment and their levels after IPGTT. Serum samples were collected from the mice in Figure 3 before IPGTT and collected again after IPGTT. ELISA was used to measure C-peptide levels of all mice in both groups. Compared to controls, C-peptide levels of anti-CD3 treated mice were significantly lower before and after IPGTT (* P
    Isotype Igg, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/isotype igg/product/BioLegend
    Average 99 stars, based on 41 article reviews
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    isotype igg - by Bioz Stars, 2019-05
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    93
    R&D Systems rat igg1 isotype control
    Specificity analyses for library-derived and designer lead IgGs by flow cytometry using transiently-transfected HEK-293 cells. Analyses of binding specificity were performed on HEK-293 cells transiently transfected with plasmids encoding either (A) human PD1 and human VEGFR2, (B) human FZD5 and human ULBP2. All plots show the target of interest transfected cells (grey line) versus ZS green marker-only transfected cells (black line). Each antibody was used in repeat staining at 5 μg/ml. These analyses confirmed that all antibodies (other than the isotype control <t>IgG1)</t> exhibited binding to PD1, but no antibody exhibited measurable signal on ZS-green, VEGFR2, FZD5 or ULBP2 transfected cells.
    Rat Igg1 Isotype Control, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rat igg1 isotype control/product/R&D Systems
    Average 93 stars, based on 107 article reviews
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    93
    Becton Dickinson isotype control igg
    Anti-CD49d antibody injections did not affect plaque load in <t>APP/PS1</t> mice. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, <t>IgG</t> isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed, serially sectioned, and immunostained using anti-Aβ (4G8) antibody. Representative images from 3-6 animals per group are shown at 20X magnification with 63X magnification insets. Quantitation of immunostaining was performed and O.D. values were averaged and plotted ± SD.
    Isotype Control Igg, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/isotype control igg/product/Becton Dickinson
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    86
    Bio X Cell mouse igg1 isotype antibody
    Inflammatory cell infiltrates in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA <t>IgG1</t> antibody. (A) Legend. (B,C) Bar graphs show mean ± SEM and individual data for inflammatory cell infiltrates (scores, U) accumulating (B) perivascularly / peribronchially or in the (C) alveoli / interstitium of the lungs. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p
    Mouse Igg1 Isotype Antibody, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 86/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse igg1 isotype antibody/product/Bio X Cell
    Average 86 stars, based on 25 article reviews
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    99
    BioLegend isotype matched control mabs
    Ligation of SIRPα impairs the reorganization of surface <t>FcγRI.</t> (A) Human macrophages were incubated for 24 h in wells coated with PLL, 20 µg/ml of hCD47, or with increasing concentrations of hCD47 in the presence of 10 µg/ml of hIgG, as indicated. M-CSF release was assessed by ELISA. Bars represent mean ± SD from three donors. Each color represents one individual donor. (B) TIRF images of FcγRI at the surface of human macrophages incubated for 10 min on slides coated with hCD47 or hCD47 plus hIgG and stained with fluorescently labeled specific antibody. Bars, 10 µm. (C) TIRF and dSTORM images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 min on slides coated with hCD47 (top) or hCD47 plus hIgG (bottom) and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 <t>mAbs.</t> Bars, 5 µm. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (D and G) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα (D) and for FcγRI and pSHP-1 Y536 (G) in cells seeded onto slides coated with PLL (light gray), hCD47 (light red), hCD47 plus hIgG (dark red), or hIgG (dark gray) for 10 (D) or 5 min (G). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (E and H) NND analysis from data shown in D and G, respectively. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P
    Isotype Matched Control Mabs, supplied by BioLegend, used in various techniques. Bioz Stars score: 99/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/isotype matched control mabs/product/BioLegend
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    81
    Agilent technologies bq16 isotype igg1 mouse
    Ligation of SIRPα impairs the reorganization of surface <t>FcγRI.</t> (A) Human macrophages were incubated for 24 h in wells coated with PLL, 20 µg/ml of hCD47, or with increasing concentrations of hCD47 in the presence of 10 µg/ml of hIgG, as indicated. M-CSF release was assessed by ELISA. Bars represent mean ± SD from three donors. Each color represents one individual donor. (B) TIRF images of FcγRI at the surface of human macrophages incubated for 10 min on slides coated with hCD47 or hCD47 plus hIgG and stained with fluorescently labeled specific antibody. Bars, 10 µm. (C) TIRF and dSTORM images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 min on slides coated with hCD47 (top) or hCD47 plus hIgG (bottom) and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 <t>mAbs.</t> Bars, 5 µm. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (D and G) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα (D) and for FcγRI and pSHP-1 Y536 (G) in cells seeded onto slides coated with PLL (light gray), hCD47 (light red), hCD47 plus hIgG (dark red), or hIgG (dark gray) for 10 (D) or 5 min (G). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (E and H) NND analysis from data shown in D and G, respectively. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P
    Bq16 Isotype Igg1 Mouse, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 81/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Zhengzhou Peptides human pdgfr alpha isotype 1
    Effect of soluble <t>PDGFR-alpha</t> on adsorption and penetration of HCMV. Virus preparations of the dual fluorescent strain TB40-BAC KL7 -UL32EGFP-UL100mCherry were preincubated with 100 ng/ml soluble Fc-chimeras for two hours and subsequently used to infect fibroblasts (HFFs) and endothelial cells (HECs) at 37°C for two hours. (A) Adsorption was assessed by counting the total number of bound virus particles per cell (pUL32-EGFP signals regardless of the pUL100-Cherry signals). Each dot represents one cell, mean values are indicated by a horizontal line, and error bars represent the standard error of the mean (SEM). Significant differences as compared to the untreated controls are indicated by asterisks. (B) The fraction of penetrated particles was determined by counting the percentage of particles lacking the envelope (particles without the pUL100-mCherry signal). Bars indicate the mean values of 15 cells per condition and error bars represent the standard error of the mean (SEM). One representative experiment out of three is shown. The significant difference between PDGFR-alpha-Fc-treated HFFs and the untreated control is indicated by an asterisk. C : Examples of microscopic images taken in HFFs. Enveloped virus particles appear yellow due to an overlap of pUL32-EGFP signals and pUL100-Cherry signals. Penetrated non-enveloped particles are only EGFP-positive. Nuclei were stained with DAPI.
    Human Pdgfr Alpha Isotype 1, supplied by Zhengzhou Peptides, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Cell Surface Shed and Secreted, Soluble HLA-G Is Endocytosed into KIR2DL4-Containing Vesicles (A) Endocytosis of soluble HLA-G in resting NK cells. The 221 cells and 221 cells transfected with HLA-Cw3 (221-Cw3) were fixed, permeabilized, and stained with mAb F4/326. Resting NK cells were incubated at 37 °C for 120 min with soluble, refolded HLA-C or HLA-G. Cells were then fixed, permeabilized, and stained with reagents to detect HLA-C (F4/326) or HLA-G (G233) as indicated. (B) The NK cell line YTS-2DL4-gfp was loaded at 37 °C for 120 min with refolded HLA-G. Cells were fixed, permeabilized, and stained with mAb G233 to detect co-localization of soluble HLA-G with gfp-tagged KIR2DL4. (C) Recombinant soluble molecules of HLA-G but not HLA-C are endocytosed into 293T-2DL4-gfp cells. Refolded HLA-G and HLA-C were incubated with 293T-2DL4-gfp cells for 2 h. Cells were then fixed, permeabilized, and stained with either mAb G233 (to detect endocytosed HLA-G; upper) or mAb F4/326 (to detect endocytosed HLA-C; middle). (D) The 293T-2DL4-gfp cells were co-cultured with an equal number of 221 cells, 221 cells expressing transmembrane HLA-G (221-G), and 221 cells expressing a soluble isoform of HLA-G (221-sG) for 48 h. Adherent 293T-2DL4-gfp cells were fixed, permeabilized, and stained with mAb G233 followed by Alexa-568–conjugated secondary antibodies prior to acquisition of confocal images. Two 221-G cells are visible in the middle panel.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: Cell Surface Shed and Secreted, Soluble HLA-G Is Endocytosed into KIR2DL4-Containing Vesicles (A) Endocytosis of soluble HLA-G in resting NK cells. The 221 cells and 221 cells transfected with HLA-Cw3 (221-Cw3) were fixed, permeabilized, and stained with mAb F4/326. Resting NK cells were incubated at 37 °C for 120 min with soluble, refolded HLA-C or HLA-G. Cells were then fixed, permeabilized, and stained with reagents to detect HLA-C (F4/326) or HLA-G (G233) as indicated. (B) The NK cell line YTS-2DL4-gfp was loaded at 37 °C for 120 min with refolded HLA-G. Cells were fixed, permeabilized, and stained with mAb G233 to detect co-localization of soluble HLA-G with gfp-tagged KIR2DL4. (C) Recombinant soluble molecules of HLA-G but not HLA-C are endocytosed into 293T-2DL4-gfp cells. Refolded HLA-G and HLA-C were incubated with 293T-2DL4-gfp cells for 2 h. Cells were then fixed, permeabilized, and stained with either mAb G233 (to detect endocytosed HLA-G; upper) or mAb F4/326 (to detect endocytosed HLA-C; middle). (D) The 293T-2DL4-gfp cells were co-cultured with an equal number of 221 cells, 221 cells expressing transmembrane HLA-G (221-G), and 221 cells expressing a soluble isoform of HLA-G (221-sG) for 48 h. Adherent 293T-2DL4-gfp cells were fixed, permeabilized, and stained with mAb G233 followed by Alexa-568–conjugated secondary antibodies prior to acquisition of confocal images. Two 221-G cells are visible in the middle panel.

    Article Snippet: Anti-HA mAb was obtained from Cell Signaling (Beverly, Massachusetts, United States), and all isotype control mAbs were obtained from Sigma (St. Louis, Missouri, United States).

    Techniques: Transfection, Staining, Incubation, Recombinant, Cell Culture, Expressing

    Binding of KIR2DL4-Ig Fusion Proteins to HLA-G–Expressing Cells Is Blocked by Anti-KIR2DL4 and Anti-HLA Class I mAbs (Top) 221-G cells were stained with mAb DX17 (pan–HLA class I mAb) and mAb G233 (HLA-G–specific mAb) (solid lines). Staining with secondary antibody alone is also shown (dotted lines). (Bottom) The 221 and 221-G cells were incubated with 50 μg/ml KIR2DL4-Ig fusion protein in the presence of 20 μg/ml of either isotype-matched control Abs or mAbs specific for class I (DX17), HLA-G (G233), or KIR2DL4 (33). Cells were then stained with goat anti-human IgG1 secondary antibodies and assessed by flow cytometry. The data are expressed as mean fluorescence intensity (MFI).

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: Binding of KIR2DL4-Ig Fusion Proteins to HLA-G–Expressing Cells Is Blocked by Anti-KIR2DL4 and Anti-HLA Class I mAbs (Top) 221-G cells were stained with mAb DX17 (pan–HLA class I mAb) and mAb G233 (HLA-G–specific mAb) (solid lines). Staining with secondary antibody alone is also shown (dotted lines). (Bottom) The 221 and 221-G cells were incubated with 50 μg/ml KIR2DL4-Ig fusion protein in the presence of 20 μg/ml of either isotype-matched control Abs or mAbs specific for class I (DX17), HLA-G (G233), or KIR2DL4 (33). Cells were then stained with goat anti-human IgG1 secondary antibodies and assessed by flow cytometry. The data are expressed as mean fluorescence intensity (MFI).

    Article Snippet: Anti-HA mAb was obtained from Cell Signaling (Beverly, Massachusetts, United States), and all isotype control mAbs were obtained from Sigma (St. Louis, Missouri, United States).

    Techniques: Binding Assay, Expressing, Staining, Incubation, Flow Cytometry, Cytometry, Fluorescence

    Endocytosis of Soluble HLA-G into 29 3T-2DL4-gfp Cells Is Blocked by Anti-KIR2DL4 mAb and by Soluble KIR2DL4 (A) Recombinant, soluble, sHLA-G at 50 μg/ml or mAb 33 (50 μg/ml) was incubated with 293T-2DL4-gfp cells. sHLA-G was also incubated together with 50 μg/ml mAb 33, 50 μg/ml KIR2DL1-Ig, or 50 μg/ml KIR2DL4-Ig, as indicated on the left. Cells were fixed, permeabilized, and stained with either Alexa-568–conjugated secondary antibodies to detect mAb 33 or anti-HLA-G mAb G233, as indicated. Individual confocal sections are shown. (B) Uptake of sHLA-G into 293T-2DL4-gfp cells correlates with level of KIR2DL4 expression. Red fluorescence intensity of G233 staining and green fluorescence intensity of gfp were quantified in 38 individual cells and plotted on a log scale. A best-fit line was generated by linear regression analysis using EXCEL data analysis software. (C) Ratio of red to green fluorescence was quantified for each loading condition as indicated. Average of 10 cells is shown, and standard deviation is shown as bars.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: Endocytosis of Soluble HLA-G into 29 3T-2DL4-gfp Cells Is Blocked by Anti-KIR2DL4 mAb and by Soluble KIR2DL4 (A) Recombinant, soluble, sHLA-G at 50 μg/ml or mAb 33 (50 μg/ml) was incubated with 293T-2DL4-gfp cells. sHLA-G was also incubated together with 50 μg/ml mAb 33, 50 μg/ml KIR2DL1-Ig, or 50 μg/ml KIR2DL4-Ig, as indicated on the left. Cells were fixed, permeabilized, and stained with either Alexa-568–conjugated secondary antibodies to detect mAb 33 or anti-HLA-G mAb G233, as indicated. Individual confocal sections are shown. (B) Uptake of sHLA-G into 293T-2DL4-gfp cells correlates with level of KIR2DL4 expression. Red fluorescence intensity of G233 staining and green fluorescence intensity of gfp were quantified in 38 individual cells and plotted on a log scale. A best-fit line was generated by linear regression analysis using EXCEL data analysis software. (C) Ratio of red to green fluorescence was quantified for each loading condition as indicated. Average of 10 cells is shown, and standard deviation is shown as bars.

    Article Snippet: Anti-HA mAb was obtained from Cell Signaling (Beverly, Massachusetts, United States), and all isotype control mAbs were obtained from Sigma (St. Louis, Missouri, United States).

    Techniques: Recombinant, Incubation, Staining, Expressing, Fluorescence, Generated, Software, Standard Deviation

    KIR2DL4 Is Endocytosed into Intracellular Vesicles (A) The B cell line 721.221 (221), resting NK cells, and activated NK cells were fixed, permeabilized, and stained with Cy3-conjugated mAb 33. In the image on the right, resting NK cells were fixed, permeabilized, and stained with a polyclonal antibody specific for the tail of KIR2DL1, followed by Alexa-568–conjugated secondary antibodies. (B) Resting NK cells were incubated at 37 °C for 30 min or 120 min with KIR2DL4-specific mAb 33-Cy3, with a Fab of control mAb 2A8 or with a Fab of anti-KIR2DL4 mAb 33, as indicated. Cells were then fixed and analyzed by confocal microscopy. (C) The 293T cells stably transfected with KIR2DL4-gfp (293T-2DL4-gfp) were fixed, permeabilized, and stained with anti-KIR2DL4 mAb 33 followed by Alexa-568–conjugated secondary antibodies to show co-localization with KIR2DL4-gfp. Single confocal sections are shown. (D) The 293T-2DL4-gfp cells were incubated at 37 °C for 120 min with anti-KIR2DL4 mAb 33, either intact (33 whole Ab) or as a Fab (33-Fab). Cells were fixed, stained with Alexa-568–conjugated secondary antibodies, and analyzed by confocal microscopy. Single confocal sections are shown.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: KIR2DL4 Is Endocytosed into Intracellular Vesicles (A) The B cell line 721.221 (221), resting NK cells, and activated NK cells were fixed, permeabilized, and stained with Cy3-conjugated mAb 33. In the image on the right, resting NK cells were fixed, permeabilized, and stained with a polyclonal antibody specific for the tail of KIR2DL1, followed by Alexa-568–conjugated secondary antibodies. (B) Resting NK cells were incubated at 37 °C for 30 min or 120 min with KIR2DL4-specific mAb 33-Cy3, with a Fab of control mAb 2A8 or with a Fab of anti-KIR2DL4 mAb 33, as indicated. Cells were then fixed and analyzed by confocal microscopy. (C) The 293T cells stably transfected with KIR2DL4-gfp (293T-2DL4-gfp) were fixed, permeabilized, and stained with anti-KIR2DL4 mAb 33 followed by Alexa-568–conjugated secondary antibodies to show co-localization with KIR2DL4-gfp. Single confocal sections are shown. (D) The 293T-2DL4-gfp cells were incubated at 37 °C for 120 min with anti-KIR2DL4 mAb 33, either intact (33 whole Ab) or as a Fab (33-Fab). Cells were fixed, stained with Alexa-568–conjugated secondary antibodies, and analyzed by confocal microscopy. Single confocal sections are shown.

    Article Snippet: Anti-HA mAb was obtained from Cell Signaling (Beverly, Massachusetts, United States), and all isotype control mAbs were obtained from Sigma (St. Louis, Missouri, United States).

    Techniques: Staining, Incubation, Confocal Microscopy, Stable Transfection, Transfection

    Immunolocalization of Endocytosed KIR2DL4 in NK Cells by Electron Microscopy (A) The NK cell lines NKL and YTS-2DL4-gfp were loaded with anti-KIR2DL4 mAb 33 for 120 min. Endocytosed receptor was detected using HRP-conjugated sheep anti-F(ab′) 2 mouse IgG. The HRP reaction product visible as a dark stain identifies the location of endocytosed KIR2DL4. Vesicular structures positive for KIR2DL4 ranged between 250 and 500 nm in size. (B) The NK cell line YTS, stably transfected with KIR2DL4-gfp (YTS-2DL4-gfp), was fixed, permeabilized, and stained with antibody to perforin followed by Alexa-568–conjugated secondary antibodies. Single confocal sections are shown.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: Immunolocalization of Endocytosed KIR2DL4 in NK Cells by Electron Microscopy (A) The NK cell lines NKL and YTS-2DL4-gfp were loaded with anti-KIR2DL4 mAb 33 for 120 min. Endocytosed receptor was detected using HRP-conjugated sheep anti-F(ab′) 2 mouse IgG. The HRP reaction product visible as a dark stain identifies the location of endocytosed KIR2DL4. Vesicular structures positive for KIR2DL4 ranged between 250 and 500 nm in size. (B) The NK cell line YTS, stably transfected with KIR2DL4-gfp (YTS-2DL4-gfp), was fixed, permeabilized, and stained with antibody to perforin followed by Alexa-568–conjugated secondary antibodies. Single confocal sections are shown.

    Article Snippet: Anti-HA mAb was obtained from Cell Signaling (Beverly, Massachusetts, United States), and all isotype control mAbs were obtained from Sigma (St. Louis, Missouri, United States).

    Techniques: Electron Microscopy, Staining, Stable Transfection, Transfection

    IL-8 Secretion Induced by KIR2DL4 Is Independent of the Transmembrane Arginine Residue and Requires Trafficking to Intracellular Vesicles (A) Expression of KIR2DL4 in 293T cells induces IL-8 secretion. The 293T cells were transfected with HA-tagged 2B4, gp49B, and KIR2DL4. After 48 h, culture supernatants were tested for IL-8 by ELISA. (B) Schematic representation of KIR2DL4 variants used in this study. Receptor localization was determined by confocal analysis of immunofluorescence staining of 293T cells transfected with the indicated constructs for 48 h. (C) IL-8 secretion induced by KIR2DL4 mutants in 293T cells. The 293T cells were transfected with HA-tagged KIR2DL4, KIR2DL4(RY-GT), and KIR2DL4-TR. After 48 h, culture supernatants were tested for IL-8 by ELISA. Transfection efficiency was verified by monitoring HA-positive cells by confocal microscopy. (D) Cell surface–expressed gp49B/2DL4 chimera does not induce IL-8 secretion. The 293T cells were transfected with HA-tagged 2B4, KIR2DL4, and gp49B/2DL4 chimera, as indicated. After 48 h, beads coated with control IgG (solid bars) or anti-HA mAb (hatched bars) were added at four beads per cell. Twelve hours later, culture supernatants were tested for IL-8 secretion. The percentage of 293T cells expressing receptor at the cell surface was monitored by HA staining and flow cytometry, and was as follows: vector control, 2%; 2B4, 39%; KIR2DL4, 9%; gp49B, 31%; gp49B/2DL4, 26%.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: IL-8 Secretion Induced by KIR2DL4 Is Independent of the Transmembrane Arginine Residue and Requires Trafficking to Intracellular Vesicles (A) Expression of KIR2DL4 in 293T cells induces IL-8 secretion. The 293T cells were transfected with HA-tagged 2B4, gp49B, and KIR2DL4. After 48 h, culture supernatants were tested for IL-8 by ELISA. (B) Schematic representation of KIR2DL4 variants used in this study. Receptor localization was determined by confocal analysis of immunofluorescence staining of 293T cells transfected with the indicated constructs for 48 h. (C) IL-8 secretion induced by KIR2DL4 mutants in 293T cells. The 293T cells were transfected with HA-tagged KIR2DL4, KIR2DL4(RY-GT), and KIR2DL4-TR. After 48 h, culture supernatants were tested for IL-8 by ELISA. Transfection efficiency was verified by monitoring HA-positive cells by confocal microscopy. (D) Cell surface–expressed gp49B/2DL4 chimera does not induce IL-8 secretion. The 293T cells were transfected with HA-tagged 2B4, KIR2DL4, and gp49B/2DL4 chimera, as indicated. After 48 h, beads coated with control IgG (solid bars) or anti-HA mAb (hatched bars) were added at four beads per cell. Twelve hours later, culture supernatants were tested for IL-8 secretion. The percentage of 293T cells expressing receptor at the cell surface was monitored by HA staining and flow cytometry, and was as follows: vector control, 2%; 2B4, 39%; KIR2DL4, 9%; gp49B, 31%; gp49B/2DL4, 26%.

    Article Snippet: Anti-HA mAb was obtained from Cell Signaling (Beverly, Massachusetts, United States), and all isotype control mAbs were obtained from Sigma (St. Louis, Missouri, United States).

    Techniques: Expressing, Transfection, Hemagglutination Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Construct, Confocal Microscopy, Flow Cytometry, Cytometry, Plasmid Preparation

    KIR2DL4 Resides in Endocytic Compartments Resting NK cells and 293T-2DL4-gfp cells were fixed, permeabilized, and stained with antibodies against Rab5, EEA-1, and M6PR, followed by Alexa-568–conjugated secondary antibodies. NK cells were further stained with mAb 33 coupled to Alexa-488 to detect KIR2DL4.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: KIR2DL4 Resides in Endocytic Compartments Resting NK cells and 293T-2DL4-gfp cells were fixed, permeabilized, and stained with antibodies against Rab5, EEA-1, and M6PR, followed by Alexa-568–conjugated secondary antibodies. NK cells were further stained with mAb 33 coupled to Alexa-488 to detect KIR2DL4.

    Article Snippet: Anti-HA mAb was obtained from Cell Signaling (Beverly, Massachusetts, United States), and all isotype control mAbs were obtained from Sigma (St. Louis, Missouri, United States).

    Techniques: Staining

    IFN-γ Secretion by Resting NK Cells Is Induced by Soluble Antibodies to KIR2DL4 (A) KIR2DL4 expression on the surface of NK cells immediately after isolation (day 0) or after 7, 14, or 21 d in culture with autologous feeder cells and rIL-2. Cells were stained with mAb 33. (B) Resting NK cells were incubated with soluble (10 μg/ml), plate-coated (5 μg/0.1 ml/well), or bead-bound (4 beads/cell) antibodies to KIR2DL4 (mAb 33) or CD16 (mAb 3G8). After 24 h, culture supernatants were tested by ELISA for IFN-γ production.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: IFN-γ Secretion by Resting NK Cells Is Induced by Soluble Antibodies to KIR2DL4 (A) KIR2DL4 expression on the surface of NK cells immediately after isolation (day 0) or after 7, 14, or 21 d in culture with autologous feeder cells and rIL-2. Cells were stained with mAb 33. (B) Resting NK cells were incubated with soluble (10 μg/ml), plate-coated (5 μg/0.1 ml/well), or bead-bound (4 beads/cell) antibodies to KIR2DL4 (mAb 33) or CD16 (mAb 3G8). After 24 h, culture supernatants were tested by ELISA for IFN-γ production.

    Article Snippet: Anti-HA mAb was obtained from Cell Signaling (Beverly, Massachusetts, United States), and all isotype control mAbs were obtained from Sigma (St. Louis, Missouri, United States).

    Techniques: Expressing, Isolation, Staining, Incubation, Enzyme-linked Immunosorbent Assay

    KIR2DL4 Localizes to a Subset of Endosomes Containing Rab5 The 293T cells were transfected with HA-tagged KIR2DL4 and gfp-tagged versions of Rab4, Rab5, Rab7, and Rab11. Forty hours after transfection, cells were fixed and stained with anti-HA mAb, followed by Alexa-568–conjugated secondary antibodies to detect KIR2DL4. Single confocal sections are shown.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: KIR2DL4 Localizes to a Subset of Endosomes Containing Rab5 The 293T cells were transfected with HA-tagged KIR2DL4 and gfp-tagged versions of Rab4, Rab5, Rab7, and Rab11. Forty hours after transfection, cells were fixed and stained with anti-HA mAb, followed by Alexa-568–conjugated secondary antibodies to detect KIR2DL4. Single confocal sections are shown.

    Article Snippet: Anti-HA mAb was obtained from Cell Signaling (Beverly, Massachusetts, United States), and all isotype control mAbs were obtained from Sigma (St. Louis, Missouri, United States).

    Techniques: Transfection, Hemagglutination Assay, Staining

    Cytokine/Chemokine Synthesis Induced by Soluble HLA-G in Resting NK Cells Resting NK cells (5 × 10 5 cells/well) from three different donors were incubated separately for 48 h with either soluble, control IgM Ab (cIg), soluble anti-KIR2DL4 IgM mAb 36 (anti-2DL4), soluble HLA-G produced in CHO cells (sHLA-G), or beads coated with control anti-HA IgG1 mAb 16B12 (cIg) or with anti-CD16 IgG1 mAb 3G8 (3G8), as indicated. sHLA-G was used together with control IgG2a mAb (cIg) or with anti-HLA-G IgG2a mAb G233 (G233), as indicated. Secretion of the cytokines/chemokines listed on the left is given in pg/ml for each donor separately. Secretion induced by sHLA-G and by Ab-coated beads was compared to that induced by anti-KIR2DL4 mAb for each donor separately and is expressed as a percentage of the anti-KIR2DL4 response. The graphs represent the average ± standard deviation from three experiments.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: Cytokine/Chemokine Synthesis Induced by Soluble HLA-G in Resting NK Cells Resting NK cells (5 × 10 5 cells/well) from three different donors were incubated separately for 48 h with either soluble, control IgM Ab (cIg), soluble anti-KIR2DL4 IgM mAb 36 (anti-2DL4), soluble HLA-G produced in CHO cells (sHLA-G), or beads coated with control anti-HA IgG1 mAb 16B12 (cIg) or with anti-CD16 IgG1 mAb 3G8 (3G8), as indicated. sHLA-G was used together with control IgG2a mAb (cIg) or with anti-HLA-G IgG2a mAb G233 (G233), as indicated. Secretion of the cytokines/chemokines listed on the left is given in pg/ml for each donor separately. Secretion induced by sHLA-G and by Ab-coated beads was compared to that induced by anti-KIR2DL4 mAb for each donor separately and is expressed as a percentage of the anti-KIR2DL4 response. The graphs represent the average ± standard deviation from three experiments.

    Article Snippet: Anti-HA mAb was obtained from Cell Signaling (Beverly, Massachusetts, United States), and all isotype control mAbs were obtained from Sigma (St. Louis, Missouri, United States).

    Techniques: Incubation, Produced, Hemagglutination Assay, Standard Deviation

    Binding of Soluble mAb to KIR2DL4 Up-regulates Multiple Genes in Resting NK Cells (A) All genes exhibiting greater than 2-fold up-regulation in microarray experiments with resting NK cells of at least two of three different individuals are listed. (B) Semiquantitative RT-PCR was performed on total RNA isolated at different time points from resting NK cells stimulated with either control IgM mAbs or anti-KIR2DL4 IgM mAbs 36 and 64. (C) Time course of TNF-α, IL-1β, and IFN-γ secretion by resting NK cells stimulated with anti-KIR2DL4 mAbs 36 and 64 (open symbols) or control IgM mAbs (closed symbols). Squares and triangles represent data obtained from two different donors. Protein secretion was detected by ELISA.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: Binding of Soluble mAb to KIR2DL4 Up-regulates Multiple Genes in Resting NK Cells (A) All genes exhibiting greater than 2-fold up-regulation in microarray experiments with resting NK cells of at least two of three different individuals are listed. (B) Semiquantitative RT-PCR was performed on total RNA isolated at different time points from resting NK cells stimulated with either control IgM mAbs or anti-KIR2DL4 IgM mAbs 36 and 64. (C) Time course of TNF-α, IL-1β, and IFN-γ secretion by resting NK cells stimulated with anti-KIR2DL4 mAbs 36 and 64 (open symbols) or control IgM mAbs (closed symbols). Squares and triangles represent data obtained from two different donors. Protein secretion was detected by ELISA.

    Article Snippet: Anti-HA mAb was obtained from Cell Signaling (Beverly, Massachusetts, United States), and all isotype control mAbs were obtained from Sigma (St. Louis, Missouri, United States).

    Techniques: Binding Assay, Microarray, Reverse Transcription Polymerase Chain Reaction, Isolation, Enzyme-linked Immunosorbent Assay

    Internalization of KIR2DL4 Is Dynamin Dependent The 293T cells transfected with HA-tagged KIR2DL4 together with either wild-type dynamin-gfp (Dynamin Egfp-WT) or a dominant-negative mutant of dynamin (dynamin Egfp-K44A) were loaded with KIR2DL4-specific Cy3-conjugated mAb 33 for 120 min and fixed. Individual confocal sections are shown.

    Journal: PLoS Biology

    Article Title: Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-GInside a Killer: Immune Signals May Promote Vascular Growth

    doi: 10.1371/journal.pbio.0040009

    Figure Lengend Snippet: Internalization of KIR2DL4 Is Dynamin Dependent The 293T cells transfected with HA-tagged KIR2DL4 together with either wild-type dynamin-gfp (Dynamin Egfp-WT) or a dominant-negative mutant of dynamin (dynamin Egfp-K44A) were loaded with KIR2DL4-specific Cy3-conjugated mAb 33 for 120 min and fixed. Individual confocal sections are shown.

    Article Snippet: Anti-HA mAb was obtained from Cell Signaling (Beverly, Massachusetts, United States), and all isotype control mAbs were obtained from Sigma (St. Louis, Missouri, United States).

    Techniques: Transfection, Hemagglutination Assay, Dominant Negative Mutation, Mutagenesis

    Inflammatory cell infiltrates in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B,C) Bar graphs show mean ± SEM and individual data for inflammatory cell infiltrates (scores, U) accumulating (B) perivascularly / peribronchially or in the (C) alveoli / interstitium of the lungs. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Journal: PLoS ONE

    Article Title: The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

    doi: 10.1371/journal.pone.0129910

    Figure Lengend Snippet: Inflammatory cell infiltrates in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B,C) Bar graphs show mean ± SEM and individual data for inflammatory cell infiltrates (scores, U) accumulating (B) perivascularly / peribronchially or in the (C) alveoli / interstitium of the lungs. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Article Snippet: IgG1-isotype control antibodies used to set the gates were from ebioscience or biolegend.

    Techniques: Gene Knockout, Mouse Assay, Injection

    IL-13 IL-17A response to exposure with OVA-PM 2.5 in wild type and B cell KO mice. Lymph node and lung tissues from groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B) Representative dot plots were generated by flow cytometry of CD4+ T cells (CD3-CD4-dual-positive) showing staining for IL-13 vs. IL-17A. (C-E) The flow cytometry data were numerically analyzed to calculate the numbers for each cell type (mean ± SEM) per lung draining lymph node. (F-H) Gene expression in the lungs of IL-13, IL-17A, IL-17F is indicated (mean ± SEM) as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Journal: PLoS ONE

    Article Title: The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

    doi: 10.1371/journal.pone.0129910

    Figure Lengend Snippet: IL-13 IL-17A response to exposure with OVA-PM 2.5 in wild type and B cell KO mice. Lymph node and lung tissues from groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B) Representative dot plots were generated by flow cytometry of CD4+ T cells (CD3-CD4-dual-positive) showing staining for IL-13 vs. IL-17A. (C-E) The flow cytometry data were numerically analyzed to calculate the numbers for each cell type (mean ± SEM) per lung draining lymph node. (F-H) Gene expression in the lungs of IL-13, IL-17A, IL-17F is indicated (mean ± SEM) as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Article Snippet: IgG1-isotype control antibodies used to set the gates were from ebioscience or biolegend.

    Techniques: Gene Knockout, Mouse Assay, Injection, Generated, Flow Cytometry, Cytometry, Staining, Expressing

    Right ventricular systolic pressures in wild type and B cell KO mice. Wild type and B cell KO mice were challenged with saline, or antigen and PM 2.5 (OVA-PM 2.5 ) intranasally. Data were pooled from 3 experiments; circles represent the data from individual mice, n = 7–8 per group. OVA-PM 2.5 challenged B cell KO control mice were injected with control antibody (open circles) or given no injections (filled circles). Another group of B cell KO mice was injected with antigen specific IgG1 (anti-OVA monoclonal). Right ventricular systolic pressure (RVSP, mmHg) data are shown as box plot of the medians (A) or as bar graph of the numbers of mice with RVSP less or greater than 26 mmHg (B) . The left-most beginning of each horizontal line indicates the group with which pairwise comparisons were made using the Mann-Whitney U test (A) or Fisher’s exact test (B) . Significant P values (P

    Journal: PLoS ONE

    Article Title: The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

    doi: 10.1371/journal.pone.0129910

    Figure Lengend Snippet: Right ventricular systolic pressures in wild type and B cell KO mice. Wild type and B cell KO mice were challenged with saline, or antigen and PM 2.5 (OVA-PM 2.5 ) intranasally. Data were pooled from 3 experiments; circles represent the data from individual mice, n = 7–8 per group. OVA-PM 2.5 challenged B cell KO control mice were injected with control antibody (open circles) or given no injections (filled circles). Another group of B cell KO mice was injected with antigen specific IgG1 (anti-OVA monoclonal). Right ventricular systolic pressure (RVSP, mmHg) data are shown as box plot of the medians (A) or as bar graph of the numbers of mice with RVSP less or greater than 26 mmHg (B) . The left-most beginning of each horizontal line indicates the group with which pairwise comparisons were made using the Mann-Whitney U test (A) or Fisher’s exact test (B) . Significant P values (P

    Article Snippet: IgG1-isotype control antibodies used to set the gates were from ebioscience or biolegend.

    Techniques: Gene Knockout, Mouse Assay, Injection, MANN-WHITNEY

    Markers of Th17 inflammation in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B-E) Bar graphs show mean ± SEM and individual data points for (B) numbers of BAL neutrophils; (C) S100a8; (D) S100a9; and (E) IL-6 gene expression. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Journal: PLoS ONE

    Article Title: The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

    doi: 10.1371/journal.pone.0129910

    Figure Lengend Snippet: Markers of Th17 inflammation in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B-E) Bar graphs show mean ± SEM and individual data points for (B) numbers of BAL neutrophils; (C) S100a8; (D) S100a9; and (E) IL-6 gene expression. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Article Snippet: IgG1-isotype control antibodies used to set the gates were from ebioscience or biolegend.

    Techniques: Gene Knockout, Mouse Assay, Injection, Expressing

    Right ventricular (RV) weight and RV-gene expression. Groups of wild type and B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B-G) Bar graphs show mean ± SEM and individual data for right ventricular weight calculated relative to (B) the weight of the left ventricle and septum (RV/LV+S), or (C) body weight (RV/BW); and gene expression in the right ventricle of [BNP (D) , IL-33 (E) , RELMα (F) , RELMγ (G) ]. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Journal: PLoS ONE

    Article Title: The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

    doi: 10.1371/journal.pone.0129910

    Figure Lengend Snippet: Right ventricular (RV) weight and RV-gene expression. Groups of wild type and B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B-G) Bar graphs show mean ± SEM and individual data for right ventricular weight calculated relative to (B) the weight of the left ventricle and septum (RV/LV+S), or (C) body weight (RV/BW); and gene expression in the right ventricle of [BNP (D) , IL-33 (E) , RELMα (F) , RELMγ (G) ]. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Article Snippet: IgG1-isotype control antibodies used to set the gates were from ebioscience or biolegend.

    Techniques: Expressing, Gene Knockout, Mouse Assay, Injection

    OVA-specific IgG1 serum levels in sensitized wild type mice and in B cell KO mice following reconstitution with monoclonal anti-OVA IgG1 antibody. OVA-specific IgG1 levels were measured in sera of sensitized wild type mice that were either challenged intranasally with saline or OVA-PM, and in sera of sensitized B cell KO mice that were either controls or injected with anti-OVA IgG1 antibody. (A) Correlation of IgG1 serum titers with right ventricular systolic pressures in sensitized wild type mice. Values for P and rs (tie corrected, Spearman’s rank correlation test) are indicated. (B) Bar graphs show (mean ± SEM) and individual data points of OVA-specific IgG1 levels. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Journal: PLoS ONE

    Article Title: The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

    doi: 10.1371/journal.pone.0129910

    Figure Lengend Snippet: OVA-specific IgG1 serum levels in sensitized wild type mice and in B cell KO mice following reconstitution with monoclonal anti-OVA IgG1 antibody. OVA-specific IgG1 levels were measured in sera of sensitized wild type mice that were either challenged intranasally with saline or OVA-PM, and in sera of sensitized B cell KO mice that were either controls or injected with anti-OVA IgG1 antibody. (A) Correlation of IgG1 serum titers with right ventricular systolic pressures in sensitized wild type mice. Values for P and rs (tie corrected, Spearman’s rank correlation test) are indicated. (B) Bar graphs show (mean ± SEM) and individual data points of OVA-specific IgG1 levels. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Article Snippet: IgG1-isotype control antibodies used to set the gates were from ebioscience or biolegend.

    Techniques: Mouse Assay, Gene Knockout, Injection

    Markers of T (helper)h2 inflammation in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B-F) Bar graphs show mean ± SEM and individual data points for (B) numbers of bronchoalveolar lavage (BAL) eosinophils; (C) mean fluorescent intensity (MFI) of major histocompatibility complex class II (MHCII) of BAL CD11c+ cells; (D) RELMβ; (E) IL-33; and (F) miR-135a expression in the lungs. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Journal: PLoS ONE

    Article Title: The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

    doi: 10.1371/journal.pone.0129910

    Figure Lengend Snippet: Markers of T (helper)h2 inflammation in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B-F) Bar graphs show mean ± SEM and individual data points for (B) numbers of bronchoalveolar lavage (BAL) eosinophils; (C) mean fluorescent intensity (MFI) of major histocompatibility complex class II (MHCII) of BAL CD11c+ cells; (D) RELMβ; (E) IL-33; and (F) miR-135a expression in the lungs. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Article Snippet: IgG1-isotype control antibodies used to set the gates were from ebioscience or biolegend.

    Techniques: Gene Knockout, Mouse Assay, Injection, Expressing

    Severe thickening of pulmonary arteries and expression of pro-remodeling genes in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B-D) Bar graphs show mean ± SEM and individual data for (B) severe arterial thickening by histological analysis and for expression of pro-remodeling genes ( C RELMα, D MMP12) in the lungs. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Journal: PLoS ONE

    Article Title: The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

    doi: 10.1371/journal.pone.0129910

    Figure Lengend Snippet: Severe thickening of pulmonary arteries and expression of pro-remodeling genes in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B-D) Bar graphs show mean ± SEM and individual data for (B) severe arterial thickening by histological analysis and for expression of pro-remodeling genes ( C RELMα, D MMP12) in the lungs. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Article Snippet: IgG1-isotype control antibodies used to set the gates were from ebioscience or biolegend.

    Techniques: Expressing, Gene Knockout, Mouse Assay, Injection

    Serum C-peptide levels of the mice at 48 h after anti-CD3 or isotype IgG treatment and their levels after IPGTT. Serum samples were collected from the mice in Figure 3 before IPGTT and collected again after IPGTT. ELISA was used to measure C-peptide levels of all mice in both groups. Compared to controls, C-peptide levels of anti-CD3 treated mice were significantly lower before and after IPGTT (* P

    Journal: Journal of Immunology Research

    Article Title: Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes

    doi: 10.1155/2014/326708

    Figure Lengend Snippet: Serum C-peptide levels of the mice at 48 h after anti-CD3 or isotype IgG treatment and their levels after IPGTT. Serum samples were collected from the mice in Figure 3 before IPGTT and collected again after IPGTT. ELISA was used to measure C-peptide levels of all mice in both groups. Compared to controls, C-peptide levels of anti-CD3 treated mice were significantly lower before and after IPGTT (* P

    Article Snippet: At 2 and 24 hours after the treatment, anti-CD3 treated mice were treated with neutralizing anti-TNF-α antibodies (BioLegend) or isotype IgG (BioLegend) (50 μ g/mouse).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Serum cytokines in mice treated with anti-CD3 antibody or isotype IgG. C57BL/6 mice were treated with anti-CD3 (4 mice) or isotype IgG (6 mice). Six hours later, serum samples were collected for cytokine measurement. The cytokines, IFN- γ , TNF- α , IL-1, and IL-6, were examined by Luminex and the statistic analysis data were shown in the figure.

    Journal: Journal of Immunology Research

    Article Title: Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes

    doi: 10.1155/2014/326708

    Figure Lengend Snippet: Serum cytokines in mice treated with anti-CD3 antibody or isotype IgG. C57BL/6 mice were treated with anti-CD3 (4 mice) or isotype IgG (6 mice). Six hours later, serum samples were collected for cytokine measurement. The cytokines, IFN- γ , TNF- α , IL-1, and IL-6, were examined by Luminex and the statistic analysis data were shown in the figure.

    Article Snippet: At 2 and 24 hours after the treatment, anti-CD3 treated mice were treated with neutralizing anti-TNF-α antibodies (BioLegend) or isotype IgG (BioLegend) (50 μ g/mouse).

    Techniques: Mouse Assay, Luminex

    Effect of anti-CD3 treatment on blood glucose of normal strain of mice. C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse) or isotype IgG (50 μ g/mouse). Four mice were included in each group. Blood glucose levels were measured at different time points as depicted in the figure. The blood glucose changes over 48 h observation were shown.

    Journal: Journal of Immunology Research

    Article Title: Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes

    doi: 10.1155/2014/326708

    Figure Lengend Snippet: Effect of anti-CD3 treatment on blood glucose of normal strain of mice. C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse) or isotype IgG (50 μ g/mouse). Four mice were included in each group. Blood glucose levels were measured at different time points as depicted in the figure. The blood glucose changes over 48 h observation were shown.

    Article Snippet: At 2 and 24 hours after the treatment, anti-CD3 treated mice were treated with neutralizing anti-TNF-α antibodies (BioLegend) or isotype IgG (BioLegend) (50 μ g/mouse).

    Techniques: Mouse Assay, Injection

    Anti-CD3 treatment enhances glucose intracellular transportation by the activated lymphocytes. (a) Freshly prepared spleen cells (1 × 10 7 /well) were cultured in a 24-well plate in 1 mL medium containing 10% fetal bovine serum with glucose concentration of 360 mg/dL. Anti-CD3 antibody (3 μ g/mL) or isotype IgG (3 μ g/mL) was added to the cultures. The culture wells were quadruplicated and incubated for 24 h. Glucose concentrations were measured before and 24 h after incubation. The results were reproduced in additional two independent experiments. (b) Freshly prepared spleen cells were stimulated with anti-CD3 antibody (3 μ g/mL) or isotype IgG (3 μ g/mL) for 3 hours; thereafter, different concentrations of 2-NBDG as indicated were added to the cultures for additional 2 hours. The fluorescent intensity of the cells was examined by BioTec plate reader using a laser filter with 480 nm excitation and 528 nm emission. The values of between anti-CD3 and isotype IgG were compared for each concentration of 2-NBDG (* P

    Journal: Journal of Immunology Research

    Article Title: Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes

    doi: 10.1155/2014/326708

    Figure Lengend Snippet: Anti-CD3 treatment enhances glucose intracellular transportation by the activated lymphocytes. (a) Freshly prepared spleen cells (1 × 10 7 /well) were cultured in a 24-well plate in 1 mL medium containing 10% fetal bovine serum with glucose concentration of 360 mg/dL. Anti-CD3 antibody (3 μ g/mL) or isotype IgG (3 μ g/mL) was added to the cultures. The culture wells were quadruplicated and incubated for 24 h. Glucose concentrations were measured before and 24 h after incubation. The results were reproduced in additional two independent experiments. (b) Freshly prepared spleen cells were stimulated with anti-CD3 antibody (3 μ g/mL) or isotype IgG (3 μ g/mL) for 3 hours; thereafter, different concentrations of 2-NBDG as indicated were added to the cultures for additional 2 hours. The fluorescent intensity of the cells was examined by BioTec plate reader using a laser filter with 480 nm excitation and 528 nm emission. The values of between anti-CD3 and isotype IgG were compared for each concentration of 2-NBDG (* P

    Article Snippet: At 2 and 24 hours after the treatment, anti-CD3 treated mice were treated with neutralizing anti-TNF-α antibodies (BioLegend) or isotype IgG (BioLegend) (50 μ g/mouse).

    Techniques: Cell Culture, Concentration Assay, Incubation

    Effect of anti-CD3 treatment on blood glucose in NOD-Rag −/− mice. T cell deficient NOD-Rag −/− mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse for 3 mice) and isotype IgG (50 μ g/mouse for 2 mice). Thereafter, the blood glucose level was monitored at different time points as shown in the figure.

    Journal: Journal of Immunology Research

    Article Title: Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes

    doi: 10.1155/2014/326708

    Figure Lengend Snippet: Effect of anti-CD3 treatment on blood glucose in NOD-Rag −/− mice. T cell deficient NOD-Rag −/− mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse for 3 mice) and isotype IgG (50 μ g/mouse for 2 mice). Thereafter, the blood glucose level was monitored at different time points as shown in the figure.

    Article Snippet: At 2 and 24 hours after the treatment, anti-CD3 treated mice were treated with neutralizing anti-TNF-α antibodies (BioLegend) or isotype IgG (BioLegend) (50 μ g/mouse).

    Techniques: Mouse Assay, Injection

    Effect of anti-CD3 antibody treatment on T and B cell activation in vitro and in vivo . (a) In vitro experiment . Spleen cells 1 × 10 6 /well in 24-well plate were stimulated with anti-CD3 antibodies (3 μ g/mL) or Isotype IgG (3 μ g/mL) for 24 hours. Thereafter, The cells were harvested and stained with CD4, CD8, B220, and CD69 and analyzed by flow cytometry. (b) In vivo experiment . C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibodies (50 μ g/mouse) or Isotype IgG (50 μ g/mouse). Three mice were included in each group. Twenty-four hours later, all mice were killed, and spleen cells of each individual mouse were prepared and stained for CD4, CD8, B220, and CD69. The expression of these markers was examined by flow cytometry.

    Journal: Journal of Immunology Research

    Article Title: Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes

    doi: 10.1155/2014/326708

    Figure Lengend Snippet: Effect of anti-CD3 antibody treatment on T and B cell activation in vitro and in vivo . (a) In vitro experiment . Spleen cells 1 × 10 6 /well in 24-well plate were stimulated with anti-CD3 antibodies (3 μ g/mL) or Isotype IgG (3 μ g/mL) for 24 hours. Thereafter, The cells were harvested and stained with CD4, CD8, B220, and CD69 and analyzed by flow cytometry. (b) In vivo experiment . C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibodies (50 μ g/mouse) or Isotype IgG (50 μ g/mouse). Three mice were included in each group. Twenty-four hours later, all mice were killed, and spleen cells of each individual mouse were prepared and stained for CD4, CD8, B220, and CD69. The expression of these markers was examined by flow cytometry.

    Article Snippet: At 2 and 24 hours after the treatment, anti-CD3 treated mice were treated with neutralizing anti-TNF-α antibodies (BioLegend) or isotype IgG (BioLegend) (50 μ g/mouse).

    Techniques: Activation Assay, In Vitro, In Vivo, Staining, Flow Cytometry, Cytometry, Mouse Assay, Injection, Expressing

    Intraperitoneal glucose tolerance test (IPGTT) in mice treated with anti-CD3 antibody or isotype IgG. C57BL/6 mice under the treatment of anti-CD3 antibody or isotype IgG as described elsewhere for 48 h received intraperitoneal injection of glucose (1 g/kg body weight). Thereafter, the blood glucose was monitored at 10, 30, 60, and 120 min. The glucose level before glucose injection served as the baseline level for each mouse. The results of all animals at different time points were depicted individually.

    Journal: Journal of Immunology Research

    Article Title: Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes

    doi: 10.1155/2014/326708

    Figure Lengend Snippet: Intraperitoneal glucose tolerance test (IPGTT) in mice treated with anti-CD3 antibody or isotype IgG. C57BL/6 mice under the treatment of anti-CD3 antibody or isotype IgG as described elsewhere for 48 h received intraperitoneal injection of glucose (1 g/kg body weight). Thereafter, the blood glucose was monitored at 10, 30, 60, and 120 min. The glucose level before glucose injection served as the baseline level for each mouse. The results of all animals at different time points were depicted individually.

    Article Snippet: At 2 and 24 hours after the treatment, anti-CD3 treated mice were treated with neutralizing anti-TNF-α antibodies (BioLegend) or isotype IgG (BioLegend) (50 μ g/mouse).

    Techniques: Mouse Assay, Injection

    The influence of cytokines on blood glucose. (a) C57BL/6 mice were treated with intraperitoneal injection of a high dose of IFN- γ (200 ng/mouse) or PBS. Thereafter, the blood glucose level was monitored at different time points as shown in the figure; (b) C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse) plus anti-IFN- γ antibody (50 μ g/mouse), or anti-CD3 antibody (50 μ g/mouse) plus isotype IgG (50 μ g/mouse). Thereafter, the blood glucose level was monitored at different time points as shown in the figure; (c) C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse) plus anti-IL-6 antibody (50 μ g/mouse), or anti-CD3 antibody (50 μ g/mouse) plus isotype IgG (50 μ g/mouse). Thereafter, the blood glucose level was monitored at different time points as shown in the figure; (d) C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse for 8 mice) or isotype IgG (50 μ g/mouse for 4 mice). Anti-CD3 group was further divided into two subgroups with one group (4 mice) receiving anti-TNF- α (50 μ g/mouse) and the other group (4 mice) receiving isotype IgG (50 μ g/mouse) at 0 and 24 h time points. Additionally, two control mice without any treatment were included in this experiment. Thereafter, the blood glucose level was monitored at different time points as shown in the figure.

    Journal: Journal of Immunology Research

    Article Title: Anti-CD3 Antibody Treatment Induces Hypoglycemia and Super Tolerance to Glucose Challenge in Mice through Enhancing Glucose Consumption by Activated Lymphocytes

    doi: 10.1155/2014/326708

    Figure Lengend Snippet: The influence of cytokines on blood glucose. (a) C57BL/6 mice were treated with intraperitoneal injection of a high dose of IFN- γ (200 ng/mouse) or PBS. Thereafter, the blood glucose level was monitored at different time points as shown in the figure; (b) C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse) plus anti-IFN- γ antibody (50 μ g/mouse), or anti-CD3 antibody (50 μ g/mouse) plus isotype IgG (50 μ g/mouse). Thereafter, the blood glucose level was monitored at different time points as shown in the figure; (c) C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse) plus anti-IL-6 antibody (50 μ g/mouse), or anti-CD3 antibody (50 μ g/mouse) plus isotype IgG (50 μ g/mouse). Thereafter, the blood glucose level was monitored at different time points as shown in the figure; (d) C57BL/6 mice were treated with intraperitoneal injection of anti-CD3 antibody (50 μ g/mouse for 8 mice) or isotype IgG (50 μ g/mouse for 4 mice). Anti-CD3 group was further divided into two subgroups with one group (4 mice) receiving anti-TNF- α (50 μ g/mouse) and the other group (4 mice) receiving isotype IgG (50 μ g/mouse) at 0 and 24 h time points. Additionally, two control mice without any treatment were included in this experiment. Thereafter, the blood glucose level was monitored at different time points as shown in the figure.

    Article Snippet: At 2 and 24 hours after the treatment, anti-CD3 treated mice were treated with neutralizing anti-TNF-α antibodies (BioLegend) or isotype IgG (BioLegend) (50 μ g/mouse).

    Techniques: Mouse Assay, Injection

    Specificity analyses for library-derived and designer lead IgGs by flow cytometry using transiently-transfected HEK-293 cells. Analyses of binding specificity were performed on HEK-293 cells transiently transfected with plasmids encoding either (A) human PD1 and human VEGFR2, (B) human FZD5 and human ULBP2. All plots show the target of interest transfected cells (grey line) versus ZS green marker-only transfected cells (black line). Each antibody was used in repeat staining at 5 μg/ml. These analyses confirmed that all antibodies (other than the isotype control IgG1) exhibited binding to PD1, but no antibody exhibited measurable signal on ZS-green, VEGFR2, FZD5 or ULBP2 transfected cells.

    Journal: mAbs

    Article Title: Anti-PD1 ‘SHR-1210ʹ aberrantly targets pro-angiogenic receptors and this polyspecificity can be ablated by paratope refinement

    doi: 10.1080/19420862.2018.1550321

    Figure Lengend Snippet: Specificity analyses for library-derived and designer lead IgGs by flow cytometry using transiently-transfected HEK-293 cells. Analyses of binding specificity were performed on HEK-293 cells transiently transfected with plasmids encoding either (A) human PD1 and human VEGFR2, (B) human FZD5 and human ULBP2. All plots show the target of interest transfected cells (grey line) versus ZS green marker-only transfected cells (black line). Each antibody was used in repeat staining at 5 μg/ml. These analyses confirmed that all antibodies (other than the isotype control IgG1) exhibited binding to PD1, but no antibody exhibited measurable signal on ZS-green, VEGFR2, FZD5 or ULBP2 transfected cells.

    Article Snippet: Mab005, SHR-1210, MAB04, MAB08, 06D02 and 12H04 in IgG1null form were also tested across a broad concentration range.

    Techniques: Derivative Assay, Flow Cytometry, Cytometry, Transfection, Binding Assay, Marker, Staining

    Specificity analyses by flow cytometry using transiently-transfected HEK-293 cells. Analyses of binding specificity were performed on HEK-293 cells transiently transfected with plasmids encoding either (A) human PD1, human VEGFR2, (B) human FZD5, human ULBP2. All plots show the target of interest transfected (grey line) versus ZS green marker-only transfected cells (black line). Transfected cells were stained using Mab005-IgG1, SHR-1210-IgG1, Pembrolizumab IgG1 null analog, and isotype IgG1. Each antibody was used in repeat staining at 5 μg/ml. These analyses confirmed that all antibodies (other than the isotype control IgG1) exhibited binding to PD1, but no antibody exhibited measurable signal on ZS-green transfected cells. Both Mab005-IgG1 and SHR-1210-IgG1 also exhibited strong binding to all targets, while the isotype IgG1 and Pembrolizumab analog did not.

    Journal: mAbs

    Article Title: Anti-PD1 ‘SHR-1210ʹ aberrantly targets pro-angiogenic receptors and this polyspecificity can be ablated by paratope refinement

    doi: 10.1080/19420862.2018.1550321

    Figure Lengend Snippet: Specificity analyses by flow cytometry using transiently-transfected HEK-293 cells. Analyses of binding specificity were performed on HEK-293 cells transiently transfected with plasmids encoding either (A) human PD1, human VEGFR2, (B) human FZD5, human ULBP2. All plots show the target of interest transfected (grey line) versus ZS green marker-only transfected cells (black line). Transfected cells were stained using Mab005-IgG1, SHR-1210-IgG1, Pembrolizumab IgG1 null analog, and isotype IgG1. Each antibody was used in repeat staining at 5 μg/ml. These analyses confirmed that all antibodies (other than the isotype control IgG1) exhibited binding to PD1, but no antibody exhibited measurable signal on ZS-green transfected cells. Both Mab005-IgG1 and SHR-1210-IgG1 also exhibited strong binding to all targets, while the isotype IgG1 and Pembrolizumab analog did not.

    Article Snippet: Mab005, SHR-1210, MAB04, MAB08, 06D02 and 12H04 in IgG1null form were also tested across a broad concentration range.

    Techniques: Flow Cytometry, Cytometry, Transfection, Binding Assay, Marker, Staining

    Flow cytometric binding to human and cyno PD1+ CHO cells. SHR-1210-IgG1, lead library-derived (A) and designer (B) IgGs were examined for specific binding on CHO-K1 cells expressing human PD1. SHR-1210-IgG1, lead library-derived (C) and designer (D) IgGs were also examined for specific binding on CHO-K1 cells expressing cyno PD1. Concentration-dependent binding was observed against human and cyno PD1 for all clones, with weaker binding being observed for SHR-1210-IgG1 in each experiment.

    Journal: mAbs

    Article Title: Anti-PD1 ‘SHR-1210ʹ aberrantly targets pro-angiogenic receptors and this polyspecificity can be ablated by paratope refinement

    doi: 10.1080/19420862.2018.1550321

    Figure Lengend Snippet: Flow cytometric binding to human and cyno PD1+ CHO cells. SHR-1210-IgG1, lead library-derived (A) and designer (B) IgGs were examined for specific binding on CHO-K1 cells expressing human PD1. SHR-1210-IgG1, lead library-derived (C) and designer (D) IgGs were also examined for specific binding on CHO-K1 cells expressing cyno PD1. Concentration-dependent binding was observed against human and cyno PD1 for all clones, with weaker binding being observed for SHR-1210-IgG1 in each experiment.

    Article Snippet: Mab005, SHR-1210, MAB04, MAB08, 06D02 and 12H04 in IgG1null form were also tested across a broad concentration range.

    Techniques: Flow Cytometry, Binding Assay, Derivative Assay, Expressing, Concentration Assay, Clone Assay

    Cell-based PD1/PD-L1 antagonism assay. Analyses of antagonism of human PD1 function at the cell surface, for example lead clones IgG1-11, IgG1-14, IgG1-06D02 and IgG1-16H10 in human IgG1null format, showed that all novel clones exhibited concentration-dependent antagonistic activity, with higher relative potency in comparison to both SHR-1210-IgG1 and IgG4 nivolumab analog.

    Journal: mAbs

    Article Title: Anti-PD1 ‘SHR-1210ʹ aberrantly targets pro-angiogenic receptors and this polyspecificity can be ablated by paratope refinement

    doi: 10.1080/19420862.2018.1550321

    Figure Lengend Snippet: Cell-based PD1/PD-L1 antagonism assay. Analyses of antagonism of human PD1 function at the cell surface, for example lead clones IgG1-11, IgG1-14, IgG1-06D02 and IgG1-16H10 in human IgG1null format, showed that all novel clones exhibited concentration-dependent antagonistic activity, with higher relative potency in comparison to both SHR-1210-IgG1 and IgG4 nivolumab analog.

    Article Snippet: Mab005, SHR-1210, MAB04, MAB08, 06D02 and 12H04 in IgG1null form were also tested across a broad concentration range.

    Techniques: Clone Assay, Concentration Assay, Activity Assay

    Direct titration ELISA for library-derived IgG1null clones binding to human and cyno PD1-Fc proteins. SHR-1210-IgG1, library-derived clones (A, B) and designer clones (C-F) in human IgG1null format were titrated (in nM) in a direct binding ELISA against human and cyno PD1-Fc proteins.

    Journal: mAbs

    Article Title: Anti-PD1 ‘SHR-1210ʹ aberrantly targets pro-angiogenic receptors and this polyspecificity can be ablated by paratope refinement

    doi: 10.1080/19420862.2018.1550321

    Figure Lengend Snippet: Direct titration ELISA for library-derived IgG1null clones binding to human and cyno PD1-Fc proteins. SHR-1210-IgG1, library-derived clones (A, B) and designer clones (C-F) in human IgG1null format were titrated (in nM) in a direct binding ELISA against human and cyno PD1-Fc proteins.

    Article Snippet: Mab005, SHR-1210, MAB04, MAB08, 06D02 and 12H04 in IgG1null form were also tested across a broad concentration range.

    Techniques: Titration, Enzyme-linked Immunosorbent Assay, Derivative Assay, Clone Assay, Binding Assay, FACS

    Off-target binding analysis ELISA assay. SHR-1210-IgG1, Mab005-IgG1, library-derived clones and designer clones in human IgG1null format were titrated (in μg/ml) in a direct binding ELISA against human PD1 (A) and cyno PD1 (B), human VEGFR2 (C) and rhesus VEGFR2 (D), human FZD5 (E) and BSA (F) proteins. These analyses confirmed that all anti-PD1 antibodies exhibited binding to PD1, but only Mab005-IgG1 and SHR-1210-IgG1 exhibited measurable off-target binding to VEGFR2 and FZD5 proteins.

    Journal: mAbs

    Article Title: Anti-PD1 ‘SHR-1210ʹ aberrantly targets pro-angiogenic receptors and this polyspecificity can be ablated by paratope refinement

    doi: 10.1080/19420862.2018.1550321

    Figure Lengend Snippet: Off-target binding analysis ELISA assay. SHR-1210-IgG1, Mab005-IgG1, library-derived clones and designer clones in human IgG1null format were titrated (in μg/ml) in a direct binding ELISA against human PD1 (A) and cyno PD1 (B), human VEGFR2 (C) and rhesus VEGFR2 (D), human FZD5 (E) and BSA (F) proteins. These analyses confirmed that all anti-PD1 antibodies exhibited binding to PD1, but only Mab005-IgG1 and SHR-1210-IgG1 exhibited measurable off-target binding to VEGFR2 and FZD5 proteins.

    Article Snippet: Mab005, SHR-1210, MAB04, MAB08, 06D02 and 12H04 in IgG1null form were also tested across a broad concentration range.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Clone Assay

    Off-target binding analysis chip re-array assay. After performing an array-based binding screen on 4,975 human receptors for SHR-1210-IgG1, confirmatory analyses of binding specificity were performed on chips in which plasmids encoding PD1 and putative SHR-1210 off-target binding proteins were arrayed and used to transfect HEK-293 cells. Effective transfection of all plasmids was confirmed by screening for the co-encoded marker ZS green (A). Separate chips were then probed in duplicate using SHR-1210-IgG1 (B), Isotype IgG1 (C), Rituximab (D), and no primary antibody (E). These analyses confirmed that only SHR-1210-IgG1 exhibited binding to PD1, but also exhibited unexpected off-target binding to VEGFR2, FZD5 and ULBP2 proteins.

    Journal: mAbs

    Article Title: Anti-PD1 ‘SHR-1210ʹ aberrantly targets pro-angiogenic receptors and this polyspecificity can be ablated by paratope refinement

    doi: 10.1080/19420862.2018.1550321

    Figure Lengend Snippet: Off-target binding analysis chip re-array assay. After performing an array-based binding screen on 4,975 human receptors for SHR-1210-IgG1, confirmatory analyses of binding specificity were performed on chips in which plasmids encoding PD1 and putative SHR-1210 off-target binding proteins were arrayed and used to transfect HEK-293 cells. Effective transfection of all plasmids was confirmed by screening for the co-encoded marker ZS green (A). Separate chips were then probed in duplicate using SHR-1210-IgG1 (B), Isotype IgG1 (C), Rituximab (D), and no primary antibody (E). These analyses confirmed that only SHR-1210-IgG1 exhibited binding to PD1, but also exhibited unexpected off-target binding to VEGFR2, FZD5 and ULBP2 proteins.

    Article Snippet: Mab005, SHR-1210, MAB04, MAB08, 06D02 and 12H04 in IgG1null form were also tested across a broad concentration range.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Transfection, Marker

    Cell-based VEGFR2 agonism assay. SHR-1210-IgG1, Mab005-IgG1, library-derived clones and designer clones in human IgG1null format were titrated (in nM) in a human VEGFR2 signalling assay. SHR-1210-IgG1, Mab005-IgG1 and the positive control protein (human VEGF-165) all induced strong, concentration-dependent VEGFR2 agonism. Lead clones IgG1-04, IgG1-08, IgG1-06D02, and IgG1-12H04 showed no measurable agonism, even at concentrations as high as 1 μM.

    Journal: mAbs

    Article Title: Anti-PD1 ‘SHR-1210ʹ aberrantly targets pro-angiogenic receptors and this polyspecificity can be ablated by paratope refinement

    doi: 10.1080/19420862.2018.1550321

    Figure Lengend Snippet: Cell-based VEGFR2 agonism assay. SHR-1210-IgG1, Mab005-IgG1, library-derived clones and designer clones in human IgG1null format were titrated (in nM) in a human VEGFR2 signalling assay. SHR-1210-IgG1, Mab005-IgG1 and the positive control protein (human VEGF-165) all induced strong, concentration-dependent VEGFR2 agonism. Lead clones IgG1-04, IgG1-08, IgG1-06D02, and IgG1-12H04 showed no measurable agonism, even at concentrations as high as 1 μM.

    Article Snippet: Mab005, SHR-1210, MAB04, MAB08, 06D02 and 12H04 in IgG1null form were also tested across a broad concentration range.

    Techniques: Derivative Assay, Clone Assay, Positive Control, Concentration Assay

    SHR-1210 epitope competition analysis of IgG1null proteins in Alphascreen. Anti-PD1 IgG1null clones were applied in an epitope competition assay using Alphascreen technology. In this assay, library-derived (A) and designer (B) IgGs were analysed for their relative affinities and retention of the parental SHR-1210 epitope by competing for SHR-1210-IgG1 binding to human PD1 protein, in solution. All clones analysed showed strong, concentration-dependent neutralization of SHR-1210-IgG1 binding to PD1, indicating maintenance of the same binding epitope.

    Journal: mAbs

    Article Title: Anti-PD1 ‘SHR-1210ʹ aberrantly targets pro-angiogenic receptors and this polyspecificity can be ablated by paratope refinement

    doi: 10.1080/19420862.2018.1550321

    Figure Lengend Snippet: SHR-1210 epitope competition analysis of IgG1null proteins in Alphascreen. Anti-PD1 IgG1null clones were applied in an epitope competition assay using Alphascreen technology. In this assay, library-derived (A) and designer (B) IgGs were analysed for their relative affinities and retention of the parental SHR-1210 epitope by competing for SHR-1210-IgG1 binding to human PD1 protein, in solution. All clones analysed showed strong, concentration-dependent neutralization of SHR-1210-IgG1 binding to PD1, indicating maintenance of the same binding epitope.

    Article Snippet: Mab005, SHR-1210, MAB04, MAB08, 06D02 and 12H04 in IgG1null form were also tested across a broad concentration range.

    Techniques: Amplified Luminescent Proximity Homogenous Assay, Clone Assay, Competitive Binding Assay, Derivative Assay, Binding Assay, Concentration Assay, Neutralization

    Off-target binding analysis re-array assay. Analyses of binding specificity were performed on chips in which plasmids encoding relevant targets were arrayed and used to transfect HEK-293 cells. Transfection of all plasmids was confirmed by screening for the co-encoded marker ZS green (A). Separate chips were then probed in duplicate using pembrolizumab analog (B), rituximab (C), Isotype IgG1 (D), SHR-1210-IgG1 (E), and example library-derived and designer lead IgGs (F-L). These analyses confirmed that all anti-PD1 antibodies exhibited binding to PD1, but only SHR-1210-IgG1 also exhibited unexpected off-target binding to VEGFR2, FZD5 and ULBP2 proteins.

    Journal: mAbs

    Article Title: Anti-PD1 ‘SHR-1210ʹ aberrantly targets pro-angiogenic receptors and this polyspecificity can be ablated by paratope refinement

    doi: 10.1080/19420862.2018.1550321

    Figure Lengend Snippet: Off-target binding analysis re-array assay. Analyses of binding specificity were performed on chips in which plasmids encoding relevant targets were arrayed and used to transfect HEK-293 cells. Transfection of all plasmids was confirmed by screening for the co-encoded marker ZS green (A). Separate chips were then probed in duplicate using pembrolizumab analog (B), rituximab (C), Isotype IgG1 (D), SHR-1210-IgG1 (E), and example library-derived and designer lead IgGs (F-L). These analyses confirmed that all anti-PD1 antibodies exhibited binding to PD1, but only SHR-1210-IgG1 also exhibited unexpected off-target binding to VEGFR2, FZD5 and ULBP2 proteins.

    Article Snippet: Mab005, SHR-1210, MAB04, MAB08, 06D02 and 12H04 in IgG1null form were also tested across a broad concentration range.

    Techniques: Binding Assay, Transfection, Marker, Derivative Assay

    Anti-CD49d antibody injections did not affect plaque load in APP/PS1 mice. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed, serially sectioned, and immunostained using anti-Aβ (4G8) antibody. Representative images from 3-6 animals per group are shown at 20X magnification with 63X magnification insets. Quantitation of immunostaining was performed and O.D. values were averaged and plotted ± SD.

    Journal: Current Alzheimer Research

    Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

    doi: 10.2174/1567205015666180801111033

    Figure Lengend Snippet: Anti-CD49d antibody injections did not affect plaque load in APP/PS1 mice. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed, serially sectioned, and immunostained using anti-Aβ (4G8) antibody. Representative images from 3-6 animals per group are shown at 20X magnification with 63X magnification insets. Quantitation of immunostaining was performed and O.D. values were averaged and plotted ± SD.

    Article Snippet: 2.4 Mice from both the strains (WT and APP/PS1) were injected intravenously (tail vein) with vehicle (saline), isotype control IgG (purified NA/LE Rat IgG2b; BD Pharmingen) antibody or 2 mg/kg purified NA/LE rat anti-mouse CD49d (BD Pharmingen, San Jose, CA) antibody once a week for 4 consecutive weeks.

    Techniques: Mouse Assay, Injection, Purification, Quantitation Assay, Immunostaining

    Anti-CD49d antibody injections altered post-synaptic proteins APP/PS1 mice. C57BL/6 and APP/PS1 mice were injected intravenous (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Parietal cortex from left brain hemispheres was dissected out, lysed and western blotted using anti-PSD95, anti-synaptophysin and anti-Cox-2 antibodies with anti-α-tubulin as loading control. Optical densities for each antibody were normalized, averaged and graphed ± SD from 3-6 animal per group (*p

    Journal: Current Alzheimer Research

    Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

    doi: 10.2174/1567205015666180801111033

    Figure Lengend Snippet: Anti-CD49d antibody injections altered post-synaptic proteins APP/PS1 mice. C57BL/6 and APP/PS1 mice were injected intravenous (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Parietal cortex from left brain hemispheres was dissected out, lysed and western blotted using anti-PSD95, anti-synaptophysin and anti-Cox-2 antibodies with anti-α-tubulin as loading control. Optical densities for each antibody were normalized, averaged and graphed ± SD from 3-6 animal per group (*p

    Article Snippet: 2.4 Mice from both the strains (WT and APP/PS1) were injected intravenously (tail vein) with vehicle (saline), isotype control IgG (purified NA/LE Rat IgG2b; BD Pharmingen) antibody or 2 mg/kg purified NA/LE rat anti-mouse CD49d (BD Pharmingen, San Jose, CA) antibody once a week for 4 consecutive weeks.

    Techniques: Mouse Assay, Injection, Purification, Western Blot

    Both the isotype control and anti-CD49d antibody injections affected brain CD4 immunoreactivity. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed and serially sectioned. The presence of IgG2b antibody in the brain was assessed by immunostaining using biotinylated anti-mouse IgG2b antibody (8A) . The presence of T cells in the brain was visualized using anti-CD4 antibody and immunoreactivity observed in the striatum (8B) and parietal cortex (8C) was imaged. Representative images from 3-6 animals per group are shown at 20X magnification with 63X magnification insets.

    Journal: Current Alzheimer Research

    Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

    doi: 10.2174/1567205015666180801111033

    Figure Lengend Snippet: Both the isotype control and anti-CD49d antibody injections affected brain CD4 immunoreactivity. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed and serially sectioned. The presence of IgG2b antibody in the brain was assessed by immunostaining using biotinylated anti-mouse IgG2b antibody (8A) . The presence of T cells in the brain was visualized using anti-CD4 antibody and immunoreactivity observed in the striatum (8B) and parietal cortex (8C) was imaged. Representative images from 3-6 animals per group are shown at 20X magnification with 63X magnification insets.

    Article Snippet: 2.4 Mice from both the strains (WT and APP/PS1) were injected intravenously (tail vein) with vehicle (saline), isotype control IgG (purified NA/LE Rat IgG2b; BD Pharmingen) antibody or 2 mg/kg purified NA/LE rat anti-mouse CD49d (BD Pharmingen, San Jose, CA) antibody once a week for 4 consecutive weeks.

    Techniques: Mouse Assay, Injection, Purification, Immunostaining

    Both the isotype control (I.C.) and anti-CD49d antibody injections reduced microgliosis in APP/PS1 mice. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed and serially sectioned. Microgliosis was assessed by immunostaining using anti-CD68 (3A) and anti-Iba-1 (3B) antibodies and total number of resting microglia were assessed using anti-TMEM119 antibody (3C) . Representative images from 3-6 animals per group are shown at 20X magnification with 63X magnification insets. Quantitation of immunostaining was performed and O.D. values were averaged and plotted ± SD.

    Journal: Current Alzheimer Research

    Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

    doi: 10.2174/1567205015666180801111033

    Figure Lengend Snippet: Both the isotype control (I.C.) and anti-CD49d antibody injections reduced microgliosis in APP/PS1 mice. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed and serially sectioned. Microgliosis was assessed by immunostaining using anti-CD68 (3A) and anti-Iba-1 (3B) antibodies and total number of resting microglia were assessed using anti-TMEM119 antibody (3C) . Representative images from 3-6 animals per group are shown at 20X magnification with 63X magnification insets. Quantitation of immunostaining was performed and O.D. values were averaged and plotted ± SD.

    Article Snippet: 2.4 Mice from both the strains (WT and APP/PS1) were injected intravenously (tail vein) with vehicle (saline), isotype control IgG (purified NA/LE Rat IgG2b; BD Pharmingen) antibody or 2 mg/kg purified NA/LE rat anti-mouse CD49d (BD Pharmingen, San Jose, CA) antibody once a week for 4 consecutive weeks.

    Techniques: Mouse Assay, Injection, Purification, Immunostaining, Quantitation Assay

    Anti-CD49d injections reduced IL-12 and IFN-γ levels in the brain. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. A portion of parietal cortex from the left brain hemispheres was dissected out, lysed, and used for multiple cytokine measurements using ELISA. Cytokine levels were determined from 3-6 animals per group ± SD (*p

    Journal: Current Alzheimer Research

    Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

    doi: 10.2174/1567205015666180801111033

    Figure Lengend Snippet: Anti-CD49d injections reduced IL-12 and IFN-γ levels in the brain. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. A portion of parietal cortex from the left brain hemispheres was dissected out, lysed, and used for multiple cytokine measurements using ELISA. Cytokine levels were determined from 3-6 animals per group ± SD (*p

    Article Snippet: 2.4 Mice from both the strains (WT and APP/PS1) were injected intravenously (tail vein) with vehicle (saline), isotype control IgG (purified NA/LE Rat IgG2b; BD Pharmingen) antibody or 2 mg/kg purified NA/LE rat anti-mouse CD49d (BD Pharmingen, San Jose, CA) antibody once a week for 4 consecutive weeks.

    Techniques: Mouse Assay, Injection, Purification, Enzyme-linked Immunosorbent Assay

    IgG and anti-CD49d injections reduced multiple cytokine levels in the spleen. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Spleens were dissected out, lysed, and used for multiple cytokine measurements using ELISAs. Cytokine levels were determined from 3-6 animals per group ± SD (*p

    Journal: Current Alzheimer Research

    Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

    doi: 10.2174/1567205015666180801111033

    Figure Lengend Snippet: IgG and anti-CD49d injections reduced multiple cytokine levels in the spleen. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Spleens were dissected out, lysed, and used for multiple cytokine measurements using ELISAs. Cytokine levels were determined from 3-6 animals per group ± SD (*p

    Article Snippet: 2.4 Mice from both the strains (WT and APP/PS1) were injected intravenously (tail vein) with vehicle (saline), isotype control IgG (purified NA/LE Rat IgG2b; BD Pharmingen) antibody or 2 mg/kg purified NA/LE rat anti-mouse CD49d (BD Pharmingen, San Jose, CA) antibody once a week for 4 consecutive weeks.

    Techniques: Mouse Assay, Injection, Purification

    Anti-CD49d injections did not alter Aβ 1-40 and Aβ 1-42 levels in the brain. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Part of the parietal cortex from left brain hemispheres was dissected out, lysed and used for soluble and insoluble Aβ 1-42 and Aβ 1-40 measurements using ELISA. Aβ levels were determined from 3-6 animals per group ± SD (*p

    Journal: Current Alzheimer Research

    Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

    doi: 10.2174/1567205015666180801111033

    Figure Lengend Snippet: Anti-CD49d injections did not alter Aβ 1-40 and Aβ 1-42 levels in the brain. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Part of the parietal cortex from left brain hemispheres was dissected out, lysed and used for soluble and insoluble Aβ 1-42 and Aβ 1-40 measurements using ELISA. Aβ levels were determined from 3-6 animals per group ± SD (*p

    Article Snippet: 2.4 Mice from both the strains (WT and APP/PS1) were injected intravenously (tail vein) with vehicle (saline), isotype control IgG (purified NA/LE Rat IgG2b; BD Pharmingen) antibody or 2 mg/kg purified NA/LE rat anti-mouse CD49d (BD Pharmingen, San Jose, CA) antibody once a week for 4 consecutive weeks.

    Techniques: Mouse Assay, Injection, Purification, Enzyme-linked Immunosorbent Assay

    Anti-CD49d injections reduced astrogliosis in APP/PS1 mice. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed and serially sectioned. Astrogliosis was assessed by immunostaining using anti-GFAP antibody (4A) . Representative images from 3-6 animals per group are shown at 20X magnification. Parietal cortex from left brain hemispheres was dissected out, lysed and western blotted using anti-GFAP antibody (4B) with anti-α-tubulin as loading control. Optical densities for each antibody were normalized, averaged and graphed ± SD from 3-6 animal per group (*p

    Journal: Current Alzheimer Research

    Article Title: Anti-α4β1 Integrin Antibodies Attenuated Brain Inflammatory Changes in a Mouse Model of Alzheimer’s Disease

    doi: 10.2174/1567205015666180801111033

    Figure Lengend Snippet: Anti-CD49d injections reduced astrogliosis in APP/PS1 mice. C57BL/6 and APP/PS1 mice were injected intravenously (tail-vein) with Saline, IgG isotype control (purified NA/LE Rat IgG2b) (I.C.) or 2 mg/kg purified NA/LE rat anti-mouse CD49d (anti-CD49d) once a week for 4 weeks. Right brain hemispheres from all the mice were fixed and serially sectioned. Astrogliosis was assessed by immunostaining using anti-GFAP antibody (4A) . Representative images from 3-6 animals per group are shown at 20X magnification. Parietal cortex from left brain hemispheres was dissected out, lysed and western blotted using anti-GFAP antibody (4B) with anti-α-tubulin as loading control. Optical densities for each antibody were normalized, averaged and graphed ± SD from 3-6 animal per group (*p

    Article Snippet: 2.4 Mice from both the strains (WT and APP/PS1) were injected intravenously (tail vein) with vehicle (saline), isotype control IgG (purified NA/LE Rat IgG2b; BD Pharmingen) antibody or 2 mg/kg purified NA/LE rat anti-mouse CD49d (BD Pharmingen, San Jose, CA) antibody once a week for 4 consecutive weeks.

    Techniques: Mouse Assay, Injection, Purification, Immunostaining, Western Blot

    Inflammatory cell infiltrates in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B,C) Bar graphs show mean ± SEM and individual data for inflammatory cell infiltrates (scores, U) accumulating (B) perivascularly / peribronchially or in the (C) alveoli / interstitium of the lungs. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Journal: PLoS ONE

    Article Title: The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

    doi: 10.1371/journal.pone.0129910

    Figure Lengend Snippet: Inflammatory cell infiltrates in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B,C) Bar graphs show mean ± SEM and individual data for inflammatory cell infiltrates (scores, U) accumulating (B) perivascularly / peribronchially or in the (C) alveoli / interstitium of the lungs. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Article Snippet: B cell KO mice were injected intraperitoneally with monoclonal mouse-anti-OVA (Sigma clone OVA-14), or mouse IgG1 isotype antibody (BioXCell) at 400 μg/dose prior to the intranasal challenges.

    Techniques: Gene Knockout, Mouse Assay, Injection

    IL-13 IL-17A response to exposure with OVA-PM 2.5 in wild type and B cell KO mice. Lymph node and lung tissues from groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B) Representative dot plots were generated by flow cytometry of CD4+ T cells (CD3-CD4-dual-positive) showing staining for IL-13 vs. IL-17A. (C-E) The flow cytometry data were numerically analyzed to calculate the numbers for each cell type (mean ± SEM) per lung draining lymph node. (F-H) Gene expression in the lungs of IL-13, IL-17A, IL-17F is indicated (mean ± SEM) as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Journal: PLoS ONE

    Article Title: The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

    doi: 10.1371/journal.pone.0129910

    Figure Lengend Snippet: IL-13 IL-17A response to exposure with OVA-PM 2.5 in wild type and B cell KO mice. Lymph node and lung tissues from groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B) Representative dot plots were generated by flow cytometry of CD4+ T cells (CD3-CD4-dual-positive) showing staining for IL-13 vs. IL-17A. (C-E) The flow cytometry data were numerically analyzed to calculate the numbers for each cell type (mean ± SEM) per lung draining lymph node. (F-H) Gene expression in the lungs of IL-13, IL-17A, IL-17F is indicated (mean ± SEM) as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Article Snippet: B cell KO mice were injected intraperitoneally with monoclonal mouse-anti-OVA (Sigma clone OVA-14), or mouse IgG1 isotype antibody (BioXCell) at 400 μg/dose prior to the intranasal challenges.

    Techniques: Gene Knockout, Mouse Assay, Injection, Generated, Flow Cytometry, Cytometry, Staining, Expressing

    Right ventricular systolic pressures in wild type and B cell KO mice. Wild type and B cell KO mice were challenged with saline, or antigen and PM 2.5 (OVA-PM 2.5 ) intranasally. Data were pooled from 3 experiments; circles represent the data from individual mice, n = 7–8 per group. OVA-PM 2.5 challenged B cell KO control mice were injected with control antibody (open circles) or given no injections (filled circles). Another group of B cell KO mice was injected with antigen specific IgG1 (anti-OVA monoclonal). Right ventricular systolic pressure (RVSP, mmHg) data are shown as box plot of the medians (A) or as bar graph of the numbers of mice with RVSP less or greater than 26 mmHg (B) . The left-most beginning of each horizontal line indicates the group with which pairwise comparisons were made using the Mann-Whitney U test (A) or Fisher’s exact test (B) . Significant P values (P

    Journal: PLoS ONE

    Article Title: The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

    doi: 10.1371/journal.pone.0129910

    Figure Lengend Snippet: Right ventricular systolic pressures in wild type and B cell KO mice. Wild type and B cell KO mice were challenged with saline, or antigen and PM 2.5 (OVA-PM 2.5 ) intranasally. Data were pooled from 3 experiments; circles represent the data from individual mice, n = 7–8 per group. OVA-PM 2.5 challenged B cell KO control mice were injected with control antibody (open circles) or given no injections (filled circles). Another group of B cell KO mice was injected with antigen specific IgG1 (anti-OVA monoclonal). Right ventricular systolic pressure (RVSP, mmHg) data are shown as box plot of the medians (A) or as bar graph of the numbers of mice with RVSP less or greater than 26 mmHg (B) . The left-most beginning of each horizontal line indicates the group with which pairwise comparisons were made using the Mann-Whitney U test (A) or Fisher’s exact test (B) . Significant P values (P

    Article Snippet: B cell KO mice were injected intraperitoneally with monoclonal mouse-anti-OVA (Sigma clone OVA-14), or mouse IgG1 isotype antibody (BioXCell) at 400 μg/dose prior to the intranasal challenges.

    Techniques: Gene Knockout, Mouse Assay, Injection, MANN-WHITNEY

    Markers of Th17 inflammation in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B-E) Bar graphs show mean ± SEM and individual data points for (B) numbers of BAL neutrophils; (C) S100a8; (D) S100a9; and (E) IL-6 gene expression. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Journal: PLoS ONE

    Article Title: The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

    doi: 10.1371/journal.pone.0129910

    Figure Lengend Snippet: Markers of Th17 inflammation in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B-E) Bar graphs show mean ± SEM and individual data points for (B) numbers of BAL neutrophils; (C) S100a8; (D) S100a9; and (E) IL-6 gene expression. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Article Snippet: B cell KO mice were injected intraperitoneally with monoclonal mouse-anti-OVA (Sigma clone OVA-14), or mouse IgG1 isotype antibody (BioXCell) at 400 μg/dose prior to the intranasal challenges.

    Techniques: Gene Knockout, Mouse Assay, Injection, Expressing

    Right ventricular (RV) weight and RV-gene expression. Groups of wild type and B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B-G) Bar graphs show mean ± SEM and individual data for right ventricular weight calculated relative to (B) the weight of the left ventricle and septum (RV/LV+S), or (C) body weight (RV/BW); and gene expression in the right ventricle of [BNP (D) , IL-33 (E) , RELMα (F) , RELMγ (G) ]. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Journal: PLoS ONE

    Article Title: The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

    doi: 10.1371/journal.pone.0129910

    Figure Lengend Snippet: Right ventricular (RV) weight and RV-gene expression. Groups of wild type and B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B-G) Bar graphs show mean ± SEM and individual data for right ventricular weight calculated relative to (B) the weight of the left ventricle and septum (RV/LV+S), or (C) body weight (RV/BW); and gene expression in the right ventricle of [BNP (D) , IL-33 (E) , RELMα (F) , RELMγ (G) ]. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Article Snippet: B cell KO mice were injected intraperitoneally with monoclonal mouse-anti-OVA (Sigma clone OVA-14), or mouse IgG1 isotype antibody (BioXCell) at 400 μg/dose prior to the intranasal challenges.

    Techniques: Expressing, Gene Knockout, Mouse Assay, Injection

    OVA-specific IgG1 serum levels in sensitized wild type mice and in B cell KO mice following reconstitution with monoclonal anti-OVA IgG1 antibody. OVA-specific IgG1 levels were measured in sera of sensitized wild type mice that were either challenged intranasally with saline or OVA-PM, and in sera of sensitized B cell KO mice that were either controls or injected with anti-OVA IgG1 antibody. (A) Correlation of IgG1 serum titers with right ventricular systolic pressures in sensitized wild type mice. Values for P and rs (tie corrected, Spearman’s rank correlation test) are indicated. (B) Bar graphs show (mean ± SEM) and individual data points of OVA-specific IgG1 levels. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Journal: PLoS ONE

    Article Title: The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

    doi: 10.1371/journal.pone.0129910

    Figure Lengend Snippet: OVA-specific IgG1 serum levels in sensitized wild type mice and in B cell KO mice following reconstitution with monoclonal anti-OVA IgG1 antibody. OVA-specific IgG1 levels were measured in sera of sensitized wild type mice that were either challenged intranasally with saline or OVA-PM, and in sera of sensitized B cell KO mice that were either controls or injected with anti-OVA IgG1 antibody. (A) Correlation of IgG1 serum titers with right ventricular systolic pressures in sensitized wild type mice. Values for P and rs (tie corrected, Spearman’s rank correlation test) are indicated. (B) Bar graphs show (mean ± SEM) and individual data points of OVA-specific IgG1 levels. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Article Snippet: B cell KO mice were injected intraperitoneally with monoclonal mouse-anti-OVA (Sigma clone OVA-14), or mouse IgG1 isotype antibody (BioXCell) at 400 μg/dose prior to the intranasal challenges.

    Techniques: Mouse Assay, Gene Knockout, Injection

    Markers of T (helper)h2 inflammation in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B-F) Bar graphs show mean ± SEM and individual data points for (B) numbers of bronchoalveolar lavage (BAL) eosinophils; (C) mean fluorescent intensity (MFI) of major histocompatibility complex class II (MHCII) of BAL CD11c+ cells; (D) RELMβ; (E) IL-33; and (F) miR-135a expression in the lungs. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Journal: PLoS ONE

    Article Title: The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

    doi: 10.1371/journal.pone.0129910

    Figure Lengend Snippet: Markers of T (helper)h2 inflammation in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B-F) Bar graphs show mean ± SEM and individual data points for (B) numbers of bronchoalveolar lavage (BAL) eosinophils; (C) mean fluorescent intensity (MFI) of major histocompatibility complex class II (MHCII) of BAL CD11c+ cells; (D) RELMβ; (E) IL-33; and (F) miR-135a expression in the lungs. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Article Snippet: B cell KO mice were injected intraperitoneally with monoclonal mouse-anti-OVA (Sigma clone OVA-14), or mouse IgG1 isotype antibody (BioXCell) at 400 μg/dose prior to the intranasal challenges.

    Techniques: Gene Knockout, Mouse Assay, Injection, Expressing

    Severe thickening of pulmonary arteries and expression of pro-remodeling genes in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B-D) Bar graphs show mean ± SEM and individual data for (B) severe arterial thickening by histological analysis and for expression of pro-remodeling genes ( C RELMα, D MMP12) in the lungs. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Journal: PLoS ONE

    Article Title: The Effects of Antigen-Specific IgG1 Antibody for the Pulmonary-Hypertension-Phenotype and B Cells for Inflammation in Mice Exposed to Antigen and Fine Particles from Air Pollution

    doi: 10.1371/journal.pone.0129910

    Figure Lengend Snippet: Severe thickening of pulmonary arteries and expression of pro-remodeling genes in the lungs. Groups of wild type or B cell KO mice challenged with saline or OVA-PM were analyzed. Groups of OVA-PM challenged B cell KO mice were either controls [injected with control antibody (open circles) or given no injections (filled circles)] or injected with anti-OVA IgG1 antibody. (A) Legend. (B-D) Bar graphs show mean ± SEM and individual data for (B) severe arterial thickening by histological analysis and for expression of pro-remodeling genes ( C RELMα, D MMP12) in the lungs. Gene expression in the lungs is shown as fold-increase over the means of the wild type saline group. Pairs of letters above the bars indicate the pairs of groups that showed significant differences (p

    Article Snippet: B cell KO mice were injected intraperitoneally with monoclonal mouse-anti-OVA (Sigma clone OVA-14), or mouse IgG1 isotype antibody (BioXCell) at 400 μg/dose prior to the intranasal challenges.

    Techniques: Expressing, Gene Knockout, Mouse Assay, Injection

    Ligation of SIRPα impairs the reorganization of surface FcγRI. (A) Human macrophages were incubated for 24 h in wells coated with PLL, 20 µg/ml of hCD47, or with increasing concentrations of hCD47 in the presence of 10 µg/ml of hIgG, as indicated. M-CSF release was assessed by ELISA. Bars represent mean ± SD from three donors. Each color represents one individual donor. (B) TIRF images of FcγRI at the surface of human macrophages incubated for 10 min on slides coated with hCD47 or hCD47 plus hIgG and stained with fluorescently labeled specific antibody. Bars, 10 µm. (C) TIRF and dSTORM images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 min on slides coated with hCD47 (top) or hCD47 plus hIgG (bottom) and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (D and G) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα (D) and for FcγRI and pSHP-1 Y536 (G) in cells seeded onto slides coated with PLL (light gray), hCD47 (light red), hCD47 plus hIgG (dark red), or hIgG (dark gray) for 10 (D) or 5 min (G). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (E and H) NND analysis from data shown in D and G, respectively. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P

    Journal: The Journal of Cell Biology

    Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

    doi: 10.1083/jcb.201608094

    Figure Lengend Snippet: Ligation of SIRPα impairs the reorganization of surface FcγRI. (A) Human macrophages were incubated for 24 h in wells coated with PLL, 20 µg/ml of hCD47, or with increasing concentrations of hCD47 in the presence of 10 µg/ml of hIgG, as indicated. M-CSF release was assessed by ELISA. Bars represent mean ± SD from three donors. Each color represents one individual donor. (B) TIRF images of FcγRI at the surface of human macrophages incubated for 10 min on slides coated with hCD47 or hCD47 plus hIgG and stained with fluorescently labeled specific antibody. Bars, 10 µm. (C) TIRF and dSTORM images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 min on slides coated with hCD47 (top) or hCD47 plus hIgG (bottom) and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (D and G) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα (D) and for FcγRI and pSHP-1 Y536 (G) in cells seeded onto slides coated with PLL (light gray), hCD47 (light red), hCD47 plus hIgG (dark red), or hIgG (dark gray) for 10 (D) or 5 min (G). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (E and H) NND analysis from data shown in D and G, respectively. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P

    Article Snippet: To assess surface expression of SIRPα and FcγRI, cells were washed and blocked with 2% FBS/PBS for 30 min at 4°C and then stained with Zombie Aqua viability dye (BioLegend), anti–CD14 mAb (clone 61D3; eBioscience) conjugated with FITC and anti–SIRPα mAb (clone SE5A5; BioLegend) or anti-FcγRI (clone 10.1; BioLegend), respectively, conjugated with Allophycocyanin (APC) or isotype-matched control mAbs (mouse IgG1 isotype control, clone MOPC-21; BioLegend; conjugated with FITC or APC) for 30 min at 4°C.

    Techniques: Ligation, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Labeling, Fluorescence

    Segregation and reorganization of FcγRI is dependent on the actin cytoskeleton and formins, but not myosin II. (A) TIRF image of FcγRI (white; bars, 20 µm) and dSTORM images (bars, 5 µm) of FcγRI (green) and SIRPα (red) at the surface of human macrophages pretreated with 1 µM latrunculin A, 0.5 µM jasplakinolide, 10 µM blebbistatin or 10 µM SMIFH2. Cells were then seeded onto slides coated with PLL (nonactivated) or hIgG for 10 min, and stained with anti-FcγRI-AF488 and anti-SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column). Bars, 1 µm. (B) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα in cells pretreated with drugs as indicated and seeded onto slides coated with PLL (gray) or hIgG (latrunculin A [Lat A], dark gray; jasplakinolide [Jasp], red; SMIFH2, green; or blebbistatin [Bleb], blue) for 10 min. Data are from a minimum of 30 cells per condition from three independent donors. Bars represent mean ± SD. (C) NND analysis from data shown in B. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P

    Journal: The Journal of Cell Biology

    Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

    doi: 10.1083/jcb.201608094

    Figure Lengend Snippet: Segregation and reorganization of FcγRI is dependent on the actin cytoskeleton and formins, but not myosin II. (A) TIRF image of FcγRI (white; bars, 20 µm) and dSTORM images (bars, 5 µm) of FcγRI (green) and SIRPα (red) at the surface of human macrophages pretreated with 1 µM latrunculin A, 0.5 µM jasplakinolide, 10 µM blebbistatin or 10 µM SMIFH2. Cells were then seeded onto slides coated with PLL (nonactivated) or hIgG for 10 min, and stained with anti-FcγRI-AF488 and anti-SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column). Bars, 1 µm. (B) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα in cells pretreated with drugs as indicated and seeded onto slides coated with PLL (gray) or hIgG (latrunculin A [Lat A], dark gray; jasplakinolide [Jasp], red; SMIFH2, green; or blebbistatin [Bleb], blue) for 10 min. Data are from a minimum of 30 cells per condition from three independent donors. Bars represent mean ± SD. (C) NND analysis from data shown in B. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P

    Article Snippet: To assess surface expression of SIRPα and FcγRI, cells were washed and blocked with 2% FBS/PBS for 30 min at 4°C and then stained with Zombie Aqua viability dye (BioLegend), anti–CD14 mAb (clone 61D3; eBioscience) conjugated with FITC and anti–SIRPα mAb (clone SE5A5; BioLegend) or anti-FcγRI (clone 10.1; BioLegend), respectively, conjugated with Allophycocyanin (APC) or isotype-matched control mAbs (mouse IgG1 isotype control, clone MOPC-21; BioLegend; conjugated with FITC or APC) for 30 min at 4°C.

    Techniques: Staining

    SIRPα and FcγRI nanoclusters are constitutively associated in nonactivated human macrophages but segregate upon activation with hIgG. (A) TIRF and dSTORM images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 min on slides coated with PLL (nonactivated, top) or hIgG (middle) and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares (middle column) are shown enlarged (right columns) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. As a positive control, macrophages seeded onto PLL-coated slides were stained with anti–FcγRI-AF488 mAb followed by anti–mouse IgG1-AF647 secondary antibody (bottom). (B) CBC histograms of the single-molecule distributions of the colocalization parameter for SIRPα and FcγRI in cells seeded onto PLL- (gray) or hIgG-coated (red) slides for 10 min or for positive control data (green). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (C) Nearest-neighbor (NN) analysis from data shown in (B). Each symbol represents the median NN of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ****, P

    Journal: The Journal of Cell Biology

    Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

    doi: 10.1083/jcb.201608094

    Figure Lengend Snippet: SIRPα and FcγRI nanoclusters are constitutively associated in nonactivated human macrophages but segregate upon activation with hIgG. (A) TIRF and dSTORM images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 min on slides coated with PLL (nonactivated, top) or hIgG (middle) and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares (middle column) are shown enlarged (right columns) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. As a positive control, macrophages seeded onto PLL-coated slides were stained with anti–FcγRI-AF488 mAb followed by anti–mouse IgG1-AF647 secondary antibody (bottom). (B) CBC histograms of the single-molecule distributions of the colocalization parameter for SIRPα and FcγRI in cells seeded onto PLL- (gray) or hIgG-coated (red) slides for 10 min or for positive control data (green). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (C) Nearest-neighbor (NN) analysis from data shown in (B). Each symbol represents the median NN of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ****, P

    Article Snippet: To assess surface expression of SIRPα and FcγRI, cells were washed and blocked with 2% FBS/PBS for 30 min at 4°C and then stained with Zombie Aqua viability dye (BioLegend), anti–CD14 mAb (clone 61D3; eBioscience) conjugated with FITC and anti–SIRPα mAb (clone SE5A5; BioLegend) or anti-FcγRI (clone 10.1; BioLegend), respectively, conjugated with Allophycocyanin (APC) or isotype-matched control mAbs (mouse IgG1 isotype control, clone MOPC-21; BioLegend; conjugated with FITC or APC) for 30 min at 4°C.

    Techniques: Activation Assay, Incubation, Staining, Fluorescence, Positive Control

    Rearrangement of macrophage surface receptors triggered by mobile hIgG. (A) TIRF images of FcγRI at the surface of human macrophages incubated for 10 min on SLBs loaded with streptavidin (nonactivating) or with streptavidin-hIgG (activating) and stained with a fluorescently labeled specific antibody. Two example images are shown for each condition. Bars, 10 µm. (B) dSTORM images of FcγRI (green) and SIRPα (red) at the surface of macrophages seeded as in A and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares are shown enlarged with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C–E) Nanocluster areas (C), density (D), and percentage of localizations in nanoclusters (E) for SIRPα and FcγRI under nonactivating (black) or hIgG-activating (gray) conditions. Each symbol represents the median of several 5 × 5 µm regions from the same cell. Horizontal lines and error bars represent mean ± SD. Data are from a minimum of 30 cells from two independent experiments. ns, not significant; *, P

    Journal: The Journal of Cell Biology

    Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

    doi: 10.1083/jcb.201608094

    Figure Lengend Snippet: Rearrangement of macrophage surface receptors triggered by mobile hIgG. (A) TIRF images of FcγRI at the surface of human macrophages incubated for 10 min on SLBs loaded with streptavidin (nonactivating) or with streptavidin-hIgG (activating) and stained with a fluorescently labeled specific antibody. Two example images are shown for each condition. Bars, 10 µm. (B) dSTORM images of FcγRI (green) and SIRPα (red) at the surface of macrophages seeded as in A and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares are shown enlarged with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C–E) Nanocluster areas (C), density (D), and percentage of localizations in nanoclusters (E) for SIRPα and FcγRI under nonactivating (black) or hIgG-activating (gray) conditions. Each symbol represents the median of several 5 × 5 µm regions from the same cell. Horizontal lines and error bars represent mean ± SD. Data are from a minimum of 30 cells from two independent experiments. ns, not significant; *, P

    Article Snippet: To assess surface expression of SIRPα and FcγRI, cells were washed and blocked with 2% FBS/PBS for 30 min at 4°C and then stained with Zombie Aqua viability dye (BioLegend), anti–CD14 mAb (clone 61D3; eBioscience) conjugated with FITC and anti–SIRPα mAb (clone SE5A5; BioLegend) or anti-FcγRI (clone 10.1; BioLegend), respectively, conjugated with Allophycocyanin (APC) or isotype-matched control mAbs (mouse IgG1 isotype control, clone MOPC-21; BioLegend; conjugated with FITC or APC) for 30 min at 4°C.

    Techniques: Incubation, Staining, Labeling, Fluorescence

    FcγRs reorganize into concentric rings upon activation. (A) TIRF images of FcγRI (top) and FcγRII (bottom) at the surface of human macrophages incubated for 10 or 30 min on slides coated with PLL (nonactivated) or hIgG and stained with fluorescently labeled specific antibodies. Bars, 10 µm. (B) TIRF and dSTORM images of FcγRI (green) and FcγRII (red) at the surface of macrophages incubated for 10 or 30 min on slides coated with PLL or hIgG and stained with anti–FcγRI-AF488 and anti–FcγRII-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and FcγRII in cells seeded onto PLL- or hIgG-coated slides for 10 (light gray and dark gray, respectively) or 30 min (light red and dark red, respectively) or for positive control data (green). The positive control data in this figure are the same as in Fig. 2 . Data are from a minimum of 10 cells from three independent donors. Bars represent mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P

    Journal: The Journal of Cell Biology

    Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

    doi: 10.1083/jcb.201608094

    Figure Lengend Snippet: FcγRs reorganize into concentric rings upon activation. (A) TIRF images of FcγRI (top) and FcγRII (bottom) at the surface of human macrophages incubated for 10 or 30 min on slides coated with PLL (nonactivated) or hIgG and stained with fluorescently labeled specific antibodies. Bars, 10 µm. (B) TIRF and dSTORM images of FcγRI (green) and FcγRII (red) at the surface of macrophages incubated for 10 or 30 min on slides coated with PLL or hIgG and stained with anti–FcγRI-AF488 and anti–FcγRII-AF647 mAbs. Bars, 5 µm. Regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and FcγRII in cells seeded onto PLL- or hIgG-coated slides for 10 (light gray and dark gray, respectively) or 30 min (light red and dark red, respectively) or for positive control data (green). The positive control data in this figure are the same as in Fig. 2 . Data are from a minimum of 10 cells from three independent donors. Bars represent mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P

    Article Snippet: To assess surface expression of SIRPα and FcγRI, cells were washed and blocked with 2% FBS/PBS for 30 min at 4°C and then stained with Zombie Aqua viability dye (BioLegend), anti–CD14 mAb (clone 61D3; eBioscience) conjugated with FITC and anti–SIRPα mAb (clone SE5A5; BioLegend) or anti-FcγRI (clone 10.1; BioLegend), respectively, conjugated with Allophycocyanin (APC) or isotype-matched control mAbs (mouse IgG1 isotype control, clone MOPC-21; BioLegend; conjugated with FITC or APC) for 30 min at 4°C.

    Techniques: Activation Assay, Incubation, Staining, Labeling, Fluorescence, Positive Control

    Src-family kinase signaling, but not Syk or PI3K signaling, is indispensable for reorganization of macrophage surfaces. (A) Immunoblots of phosphorylated AKT in nonactivated (PLL) or hIgG-activated human macrophages pretreated with vehicle (DMSO), as a control, 10 µM PP2 (left), 100 µM piceatannol (PCT; middle), or 1 µM wortmannin (Wort; right). Blots represent two independent experiments. (B) TIRF image of FcγRI (white; bars, 20 µm) and dSTORM images (bars, 5 µm) of FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated with vehicle (DMSO), PP2, PCT, or Wort, pretreated as in A. Cells were then seeded onto slides coated with PLL (nonactivated) or hIgG for 10 min and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column). Bars, 1 µm. (C) CBC histograms for FcγRI and SIRPα in cells pretreated as in A and seeded onto slides coated with PLL (gray) or hIgG (DMSO, dark gray; PP2, red; PCT, green; and Wort, blue) for 10 min, as indicated. Data are from a minimum of 30 cells from three independent donors. Bars show mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; ****, P

    Journal: The Journal of Cell Biology

    Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

    doi: 10.1083/jcb.201608094

    Figure Lengend Snippet: Src-family kinase signaling, but not Syk or PI3K signaling, is indispensable for reorganization of macrophage surfaces. (A) Immunoblots of phosphorylated AKT in nonactivated (PLL) or hIgG-activated human macrophages pretreated with vehicle (DMSO), as a control, 10 µM PP2 (left), 100 µM piceatannol (PCT; middle), or 1 µM wortmannin (Wort; right). Blots represent two independent experiments. (B) TIRF image of FcγRI (white; bars, 20 µm) and dSTORM images (bars, 5 µm) of FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated with vehicle (DMSO), PP2, PCT, or Wort, pretreated as in A. Cells were then seeded onto slides coated with PLL (nonactivated) or hIgG for 10 min and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column). Bars, 1 µm. (C) CBC histograms for FcγRI and SIRPα in cells pretreated as in A and seeded onto slides coated with PLL (gray) or hIgG (DMSO, dark gray; PP2, red; PCT, green; and Wort, blue) for 10 min, as indicated. Data are from a minimum of 30 cells from three independent donors. Bars show mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; ****, P

    Article Snippet: To assess surface expression of SIRPα and FcγRI, cells were washed and blocked with 2% FBS/PBS for 30 min at 4°C and then stained with Zombie Aqua viability dye (BioLegend), anti–CD14 mAb (clone 61D3; eBioscience) conjugated with FITC and anti–SIRPα mAb (clone SE5A5; BioLegend) or anti-FcγRI (clone 10.1; BioLegend), respectively, conjugated with Allophycocyanin (APC) or isotype-matched control mAbs (mouse IgG1 isotype control, clone MOPC-21; BioLegend; conjugated with FITC or APC) for 30 min at 4°C.

    Techniques: Western Blot, Incubation, Staining

    Specific activation of FcγRI is required for its reorganization into concentric rings and segregation from SIRPα nanoclusters. (A and B) TIRF (bars, 10 µm) and dSTORM (bars, 5 µm) images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 (A) or 30 min (B) on slides coated with hIgG1 or hIgG2 and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα in cells seeded onto hIgG1- or hIgG2-coated slides for 10 (light gray and dark gray, respectively) or 30 min (light red and dark red, respectively). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P

    Journal: The Journal of Cell Biology

    Article Title: Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

    doi: 10.1083/jcb.201608094

    Figure Lengend Snippet: Specific activation of FcγRI is required for its reorganization into concentric rings and segregation from SIRPα nanoclusters. (A and B) TIRF (bars, 10 µm) and dSTORM (bars, 5 µm) images showing FcγRI (green) and SIRPα (red) at the surface of human macrophages incubated for 10 (A) or 30 min (B) on slides coated with hIgG1 or hIgG2 and stained with anti–FcγRI-AF488 and anti–SIRPα-AF647 mAbs. In each condition, regions outlined by the white squares (middle column) are shown enlarged (right column) with relative fluorescence intensity profiles along the white lines. Bars, 1 µm. (C) CBC histograms of the single-molecule distributions of the colocalization parameter for FcγRI and SIRPα in cells seeded onto hIgG1- or hIgG2-coated slides for 10 (light gray and dark gray, respectively) or 30 min (light red and dark red, respectively). Data are from a minimum of 30 cells from three independent donors. Bars represent mean ± SD. (D) NND analysis from data shown in C. Each symbol represents the median NND of all paired single-molecule localizations from one cell. Horizontal lines and error bars represent mean ± SD. ns, not significant; **, P

    Article Snippet: To assess surface expression of SIRPα and FcγRI, cells were washed and blocked with 2% FBS/PBS for 30 min at 4°C and then stained with Zombie Aqua viability dye (BioLegend), anti–CD14 mAb (clone 61D3; eBioscience) conjugated with FITC and anti–SIRPα mAb (clone SE5A5; BioLegend) or anti-FcγRI (clone 10.1; BioLegend), respectively, conjugated with Allophycocyanin (APC) or isotype-matched control mAbs (mouse IgG1 isotype control, clone MOPC-21; BioLegend; conjugated with FITC or APC) for 30 min at 4°C.

    Techniques: Activation Assay, Incubation, Staining, Fluorescence

    Effect of soluble PDGFR-alpha on adsorption and penetration of HCMV. Virus preparations of the dual fluorescent strain TB40-BAC KL7 -UL32EGFP-UL100mCherry were preincubated with 100 ng/ml soluble Fc-chimeras for two hours and subsequently used to infect fibroblasts (HFFs) and endothelial cells (HECs) at 37°C for two hours. (A) Adsorption was assessed by counting the total number of bound virus particles per cell (pUL32-EGFP signals regardless of the pUL100-Cherry signals). Each dot represents one cell, mean values are indicated by a horizontal line, and error bars represent the standard error of the mean (SEM). Significant differences as compared to the untreated controls are indicated by asterisks. (B) The fraction of penetrated particles was determined by counting the percentage of particles lacking the envelope (particles without the pUL100-mCherry signal). Bars indicate the mean values of 15 cells per condition and error bars represent the standard error of the mean (SEM). One representative experiment out of three is shown. The significant difference between PDGFR-alpha-Fc-treated HFFs and the untreated control is indicated by an asterisk. C : Examples of microscopic images taken in HFFs. Enveloped virus particles appear yellow due to an overlap of pUL32-EGFP signals and pUL100-Cherry signals. Penetrated non-enveloped particles are only EGFP-positive. Nuclei were stained with DAPI.

    Journal: PLoS Pathogens

    Article Title: A derivative of platelet-derived growth factor receptor alpha binds to the trimer of human cytomegalovirus and inhibits entry into fibroblasts and endothelial cellsHuman cytomegalovirus glycoprotein complex gH/gL/gO uses PDGFR-α as a key for entry

    doi: 10.1371/journal.ppat.1006273

    Figure Lengend Snippet: Effect of soluble PDGFR-alpha on adsorption and penetration of HCMV. Virus preparations of the dual fluorescent strain TB40-BAC KL7 -UL32EGFP-UL100mCherry were preincubated with 100 ng/ml soluble Fc-chimeras for two hours and subsequently used to infect fibroblasts (HFFs) and endothelial cells (HECs) at 37°C for two hours. (A) Adsorption was assessed by counting the total number of bound virus particles per cell (pUL32-EGFP signals regardless of the pUL100-Cherry signals). Each dot represents one cell, mean values are indicated by a horizontal line, and error bars represent the standard error of the mean (SEM). Significant differences as compared to the untreated controls are indicated by asterisks. (B) The fraction of penetrated particles was determined by counting the percentage of particles lacking the envelope (particles without the pUL100-mCherry signal). Bars indicate the mean values of 15 cells per condition and error bars represent the standard error of the mean (SEM). One representative experiment out of three is shown. The significant difference between PDGFR-alpha-Fc-treated HFFs and the untreated control is indicated by an asterisk. C : Examples of microscopic images taken in HFFs. Enveloped virus particles appear yellow due to an overlap of pUL32-EGFP signals and pUL100-Cherry signals. Penetrated non-enveloped particles are only EGFP-positive. Nuclei were stained with DAPI.

    Article Snippet: The 40mer peptides based on the extracellular domain of human PDGFR-alpha isotype 1 were obtained from Phtdpeptides (Shanghai, China) at a purity of > 95%.

    Techniques: Adsorption, BAC Assay, Flow Cytometry, Staining

    Binding of soluble growth factor receptor-Fc chimeras to HCMV particles. Virus preparations of strain TB40/E were pretreated for two hours with 500 ng/ml PDGFR-alpha-Fc, PDGFR-beta-Fc or EGFR-Fc and then incubated with fibroblasts for 90 min on ice. Cells were fixed and stained for the viral structural protein pUL32 (red) and for the Fc-fusion part (green). Nuclei were counterstained with DAPI.

    Journal: PLoS Pathogens

    Article Title: A derivative of platelet-derived growth factor receptor alpha binds to the trimer of human cytomegalovirus and inhibits entry into fibroblasts and endothelial cellsHuman cytomegalovirus glycoprotein complex gH/gL/gO uses PDGFR-α as a key for entry

    doi: 10.1371/journal.ppat.1006273

    Figure Lengend Snippet: Binding of soluble growth factor receptor-Fc chimeras to HCMV particles. Virus preparations of strain TB40/E were pretreated for two hours with 500 ng/ml PDGFR-alpha-Fc, PDGFR-beta-Fc or EGFR-Fc and then incubated with fibroblasts for 90 min on ice. Cells were fixed and stained for the viral structural protein pUL32 (red) and for the Fc-fusion part (green). Nuclei were counterstained with DAPI.

    Article Snippet: The 40mer peptides based on the extracellular domain of human PDGFR-alpha isotype 1 were obtained from Phtdpeptides (Shanghai, China) at a purity of > 95%.

    Techniques: Binding Assay, Flow Cytometry, Incubation, Staining

    Inhibitory effect of PDGFR-alpha-derived peptides. The luciferase reporter virus TB40-BAC4-IE-Gluc was preincubated with 40mer peptides derived from the extracellular domain of PDGFR-alpha at concentrations from 0.05–50 nmol/ml for 2 h before infection of fibroblasts (HFFs) and endothelial cells (HECs). Untreated virus was used as a control for uninhibited infection. One day postinfection, supernatants were analyzed for luciferase activity indicating the degree of infection. The extent of inhibition was calculated as 1 - (luminescence with peptide / luminescence without peptide) and is given as percentage. Error bars indicate the standard error of the mean (SEM) of four data sets per peptide. The number of the peptide (#1-#17) indicates its position in the protein, starting from the N-terminus. Arrowheads indicate the concentration at which 50% of infection are inhibited.

    Journal: PLoS Pathogens

    Article Title: A derivative of platelet-derived growth factor receptor alpha binds to the trimer of human cytomegalovirus and inhibits entry into fibroblasts and endothelial cellsHuman cytomegalovirus glycoprotein complex gH/gL/gO uses PDGFR-α as a key for entry

    doi: 10.1371/journal.ppat.1006273

    Figure Lengend Snippet: Inhibitory effect of PDGFR-alpha-derived peptides. The luciferase reporter virus TB40-BAC4-IE-Gluc was preincubated with 40mer peptides derived from the extracellular domain of PDGFR-alpha at concentrations from 0.05–50 nmol/ml for 2 h before infection of fibroblasts (HFFs) and endothelial cells (HECs). Untreated virus was used as a control for uninhibited infection. One day postinfection, supernatants were analyzed for luciferase activity indicating the degree of infection. The extent of inhibition was calculated as 1 - (luminescence with peptide / luminescence without peptide) and is given as percentage. Error bars indicate the standard error of the mean (SEM) of four data sets per peptide. The number of the peptide (#1-#17) indicates its position in the protein, starting from the N-terminus. Arrowheads indicate the concentration at which 50% of infection are inhibited.

    Article Snippet: The 40mer peptides based on the extracellular domain of human PDGFR-alpha isotype 1 were obtained from Phtdpeptides (Shanghai, China) at a purity of > 95%.

    Techniques: Derivative Assay, Luciferase, Infection, Activity Assay, Inhibition, Concentration Assay

    Postadsorption-inhibitory effect of soluble PDGFR-alpha-Fc. Virus preparations were adsorbed to fibroblasts on ice for 60 min and subsequently incubated with 200 ng/ml PDGFR-alpha-Fc for two hours on ice. Cells were then either directly shifted to 37°C or first treated with the chemical fusogen PEG. After an overnight incubation, cells were fixed and stained for viral IE antigens. (A) For each condition, the fraction of infected cells (immediate-early antigen positive cells/total cells) was compared to the untreated control. Mean values of 3 independent experiments are shown. Error bars indicate the standard error of the mean (SEM). The significant difference between PDGFR-alpha-Fc-treated cells and the untreated control is indicated by an asterisk. (B) Examples of HCMV IE antigen expression (red fluorescence) after treatment with PDGFR-alpha-Fc alone or PDGFR-alpha-Fc and PEG. Nuclei were counterstained with DAPI.

    Journal: PLoS Pathogens

    Article Title: A derivative of platelet-derived growth factor receptor alpha binds to the trimer of human cytomegalovirus and inhibits entry into fibroblasts and endothelial cellsHuman cytomegalovirus glycoprotein complex gH/gL/gO uses PDGFR-α as a key for entry

    doi: 10.1371/journal.ppat.1006273

    Figure Lengend Snippet: Postadsorption-inhibitory effect of soluble PDGFR-alpha-Fc. Virus preparations were adsorbed to fibroblasts on ice for 60 min and subsequently incubated with 200 ng/ml PDGFR-alpha-Fc for two hours on ice. Cells were then either directly shifted to 37°C or first treated with the chemical fusogen PEG. After an overnight incubation, cells were fixed and stained for viral IE antigens. (A) For each condition, the fraction of infected cells (immediate-early antigen positive cells/total cells) was compared to the untreated control. Mean values of 3 independent experiments are shown. Error bars indicate the standard error of the mean (SEM). The significant difference between PDGFR-alpha-Fc-treated cells and the untreated control is indicated by an asterisk. (B) Examples of HCMV IE antigen expression (red fluorescence) after treatment with PDGFR-alpha-Fc alone or PDGFR-alpha-Fc and PEG. Nuclei were counterstained with DAPI.

    Article Snippet: The 40mer peptides based on the extracellular domain of human PDGFR-alpha isotype 1 were obtained from Phtdpeptides (Shanghai, China) at a purity of > 95%.

    Techniques: Flow Cytometry, Incubation, Staining, Infection, Expressing, Fluorescence

    Inhibitory effect of soluble PDGFR-alpha-Fc chimeras on HCMV infection. Virus preparations of strain TB40/E were pretreated for two hours with PDGFR-alpha-Fc at indicated concentrations and then added to fibroblasts (HFFs) and endothelial cells (HECs). One day after infection, cells were fixed and stained for viral IE antigens by indirect immunofluorescence. The ratio of IE antigen-positive cells per total cell number was calculated to represent the degree of infection. The graphs show the inhibition of infection in treated cultures as compared to untreated controls. Error bars represent the standard error of the mean (SEM).

    Journal: PLoS Pathogens

    Article Title: A derivative of platelet-derived growth factor receptor alpha binds to the trimer of human cytomegalovirus and inhibits entry into fibroblasts and endothelial cellsHuman cytomegalovirus glycoprotein complex gH/gL/gO uses PDGFR-α as a key for entry

    doi: 10.1371/journal.ppat.1006273

    Figure Lengend Snippet: Inhibitory effect of soluble PDGFR-alpha-Fc chimeras on HCMV infection. Virus preparations of strain TB40/E were pretreated for two hours with PDGFR-alpha-Fc at indicated concentrations and then added to fibroblasts (HFFs) and endothelial cells (HECs). One day after infection, cells were fixed and stained for viral IE antigens by indirect immunofluorescence. The ratio of IE antigen-positive cells per total cell number was calculated to represent the degree of infection. The graphs show the inhibition of infection in treated cultures as compared to untreated controls. Error bars represent the standard error of the mean (SEM).

    Article Snippet: The 40mer peptides based on the extracellular domain of human PDGFR-alpha isotype 1 were obtained from Phtdpeptides (Shanghai, China) at a purity of > 95%.

    Techniques: Flow Cytometry, Infection, Staining, Immunofluorescence, Inhibition

    PDGFR-alpha-Fc binds HCMV particles only in the presence of the gH/gL/pUL74 complex. (A) Virus preparations of strain TB40-BAC4 (wild type) or TB40-BAC4-UL74stop were pretreated with 500 ng/ml PDGFR-alpha-Fc for two hours at 37°C. Fibroblasts (HFFs) were then incubated with pretreated virus on ice followed by immunofluorescence staining for the viral structural protein pUL32 (red) and the Fc-fusion part of PDGFR-alpha-Fc (green). (B) Virus preparations were preincubated with variable concentrations of PDGFR-alpha-Fc for 2h before infection of HFFs and endothelial cells (HECs). One day postinfection, cells were fixed and stained by immunofluorescence for viral immediate early antigens. For each condition, the fraction of infected cells (immediate-early antigen positive cells/total cells) was compared to the untreated control. Mean values of 3 independent experiments are shown. Error bars indicate the standard error of the mean (SEM). Significant differences as compared to the untreated controls are indicated by asterisks.

    Journal: PLoS Pathogens

    Article Title: A derivative of platelet-derived growth factor receptor alpha binds to the trimer of human cytomegalovirus and inhibits entry into fibroblasts and endothelial cellsHuman cytomegalovirus glycoprotein complex gH/gL/gO uses PDGFR-α as a key for entry

    doi: 10.1371/journal.ppat.1006273

    Figure Lengend Snippet: PDGFR-alpha-Fc binds HCMV particles only in the presence of the gH/gL/pUL74 complex. (A) Virus preparations of strain TB40-BAC4 (wild type) or TB40-BAC4-UL74stop were pretreated with 500 ng/ml PDGFR-alpha-Fc for two hours at 37°C. Fibroblasts (HFFs) were then incubated with pretreated virus on ice followed by immunofluorescence staining for the viral structural protein pUL32 (red) and the Fc-fusion part of PDGFR-alpha-Fc (green). (B) Virus preparations were preincubated with variable concentrations of PDGFR-alpha-Fc for 2h before infection of HFFs and endothelial cells (HECs). One day postinfection, cells were fixed and stained by immunofluorescence for viral immediate early antigens. For each condition, the fraction of infected cells (immediate-early antigen positive cells/total cells) was compared to the untreated control. Mean values of 3 independent experiments are shown. Error bars indicate the standard error of the mean (SEM). Significant differences as compared to the untreated controls are indicated by asterisks.

    Article Snippet: The 40mer peptides based on the extracellular domain of human PDGFR-alpha isotype 1 were obtained from Phtdpeptides (Shanghai, China) at a purity of > 95%.

    Techniques: Flow Cytometry, Incubation, Immunofluorescence, Staining, Infection

    Quantification of PDGFR-alpha-Fc binding to HCMV particles. Virus preparations of strain TB40/E were preincubated with serial dilutions of PDGFR-alpha-Fc. Viruses were attached to fibroblasts by incubation on ice for 90 min followed by acetone fixation. Binding of PDGFR-alpha-Fc was assessed by direct immunofluorescence of the Fc-fusion part and quantification of signal intensities (maximum grey values per particle). In parallel, fibroblasts were incubated with the same mixtures at 37°C and stained for viral immediate-early antigens at one day postinfection to determine the fraction of infected cells. The intensity of the staining with PDGFR-alpha-Fc was measured for 100 particles in each condition. The same data set is provided with a linear scale and a logarithmic scale. Error bars indicate the error of the median.

    Journal: PLoS Pathogens

    Article Title: A derivative of platelet-derived growth factor receptor alpha binds to the trimer of human cytomegalovirus and inhibits entry into fibroblasts and endothelial cellsHuman cytomegalovirus glycoprotein complex gH/gL/gO uses PDGFR-α as a key for entry

    doi: 10.1371/journal.ppat.1006273

    Figure Lengend Snippet: Quantification of PDGFR-alpha-Fc binding to HCMV particles. Virus preparations of strain TB40/E were preincubated with serial dilutions of PDGFR-alpha-Fc. Viruses were attached to fibroblasts by incubation on ice for 90 min followed by acetone fixation. Binding of PDGFR-alpha-Fc was assessed by direct immunofluorescence of the Fc-fusion part and quantification of signal intensities (maximum grey values per particle). In parallel, fibroblasts were incubated with the same mixtures at 37°C and stained for viral immediate-early antigens at one day postinfection to determine the fraction of infected cells. The intensity of the staining with PDGFR-alpha-Fc was measured for 100 particles in each condition. The same data set is provided with a linear scale and a logarithmic scale. Error bars indicate the error of the median.

    Article Snippet: The 40mer peptides based on the extracellular domain of human PDGFR-alpha isotype 1 were obtained from Phtdpeptides (Shanghai, China) at a purity of > 95%.

    Techniques: Flow Cytometry, Binding Assay, Incubation, Immunofluorescence, Staining, Infection

    Effect of siRNA-mediated depletion of PDGFR-alpha on infection efficiency in fibroblasts and endothelial cells. Fibroblasts (A) and endothelial cells (B) were transfected with siRNAs targeting PDGFR-alpha. Non-targeting (NT) siRNAs and siRNAs against the viral immediate early (IE) proteins were included as controls. Two days after transfection, cells were infected with HCMV strain TB40/E, and the next day viral IE antigens were detected by indirect immunofluorescence for visualization of infected cells. The number of IE antigen-positive cells was counted and compared to the NT control. Error bars in (A) and (B) represent the standard error of the mean (SEM). Significant differences as compared to NT control are indicated by asterisks. C : Examples of HCMV IE antigen expression (red fluorescence) in HFFs after treatment with the respective siRNAs. Nuclei of non-infected cells appear blue due to counterstaining with DAPI. D: Fluorescence-activated cell sorting (FACS) for detection of PDGFR-alpha on the surface of fibroblasts and endothelial cells 2 d after treatment with PDGFR-alpha siRNA or non-targeting (NT) siRNA. NT siRNA represents PDGFR-alpha levels without specific knockdown. Isotype control antibody was included as a negative control for staining.

    Journal: PLoS Pathogens

    Article Title: A derivative of platelet-derived growth factor receptor alpha binds to the trimer of human cytomegalovirus and inhibits entry into fibroblasts and endothelial cellsHuman cytomegalovirus glycoprotein complex gH/gL/gO uses PDGFR-α as a key for entry

    doi: 10.1371/journal.ppat.1006273

    Figure Lengend Snippet: Effect of siRNA-mediated depletion of PDGFR-alpha on infection efficiency in fibroblasts and endothelial cells. Fibroblasts (A) and endothelial cells (B) were transfected with siRNAs targeting PDGFR-alpha. Non-targeting (NT) siRNAs and siRNAs against the viral immediate early (IE) proteins were included as controls. Two days after transfection, cells were infected with HCMV strain TB40/E, and the next day viral IE antigens were detected by indirect immunofluorescence for visualization of infected cells. The number of IE antigen-positive cells was counted and compared to the NT control. Error bars in (A) and (B) represent the standard error of the mean (SEM). Significant differences as compared to NT control are indicated by asterisks. C : Examples of HCMV IE antigen expression (red fluorescence) in HFFs after treatment with the respective siRNAs. Nuclei of non-infected cells appear blue due to counterstaining with DAPI. D: Fluorescence-activated cell sorting (FACS) for detection of PDGFR-alpha on the surface of fibroblasts and endothelial cells 2 d after treatment with PDGFR-alpha siRNA or non-targeting (NT) siRNA. NT siRNA represents PDGFR-alpha levels without specific knockdown. Isotype control antibody was included as a negative control for staining.

    Article Snippet: The 40mer peptides based on the extracellular domain of human PDGFR-alpha isotype 1 were obtained from Phtdpeptides (Shanghai, China) at a purity of > 95%.

    Techniques: Infection, Transfection, Immunofluorescence, Expressing, Fluorescence, FACS, Negative Control, Staining

    The inhibitory effect of soluble PDGFR-alpha is specific and affects various HCMV strains. (A) PDGFR-alpha-Fc, PDGFR-beta-Fc and EGFR-Fc were compared regarding their inhibitory potential on infection of fibroblasts (HFFs) and endothelial cells (HECs) by HCMV strain TB40/E. Virus preparations were pretreated for 2 h with the respective growth factor receptor at indicated concentrations and then added to cell cultures for 2 h, followed by a medium exchange and incubation overnight. Cells were fixed and stained for viral IE antigens. The percentage of infection was calculated as the ratio of IE antigen-positive cells / total cell number. (B) The potential of PDGFR-alpha-Fc to inhibit fibroblast infection with various HCMV strains was tested using a collection of strains representing the known glycoprotein variants. The virus preparations were either preincubated with medium (no drug) or medium containing 250 ng/ml PDGFR-alpha-Fc. Error bars in (A) and (B) represent the standard error of the mean (SEM). The significant difference of VR1814 as compared to the other strains is indicated by an asterisk.

    Journal: PLoS Pathogens

    Article Title: A derivative of platelet-derived growth factor receptor alpha binds to the trimer of human cytomegalovirus and inhibits entry into fibroblasts and endothelial cellsHuman cytomegalovirus glycoprotein complex gH/gL/gO uses PDGFR-α as a key for entry

    doi: 10.1371/journal.ppat.1006273

    Figure Lengend Snippet: The inhibitory effect of soluble PDGFR-alpha is specific and affects various HCMV strains. (A) PDGFR-alpha-Fc, PDGFR-beta-Fc and EGFR-Fc were compared regarding their inhibitory potential on infection of fibroblasts (HFFs) and endothelial cells (HECs) by HCMV strain TB40/E. Virus preparations were pretreated for 2 h with the respective growth factor receptor at indicated concentrations and then added to cell cultures for 2 h, followed by a medium exchange and incubation overnight. Cells were fixed and stained for viral IE antigens. The percentage of infection was calculated as the ratio of IE antigen-positive cells / total cell number. (B) The potential of PDGFR-alpha-Fc to inhibit fibroblast infection with various HCMV strains was tested using a collection of strains representing the known glycoprotein variants. The virus preparations were either preincubated with medium (no drug) or medium containing 250 ng/ml PDGFR-alpha-Fc. Error bars in (A) and (B) represent the standard error of the mean (SEM). The significant difference of VR1814 as compared to the other strains is indicated by an asterisk.

    Article Snippet: The 40mer peptides based on the extracellular domain of human PDGFR-alpha isotype 1 were obtained from Phtdpeptides (Shanghai, China) at a purity of > 95%.

    Techniques: Flow Cytometry, Infection, Incubation, Staining