human cd86  (Sino Biological)


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    Name:
    Human CD86 B7 2 Protein His Tag
    Description:
    A DNA sequence encoding the extracellular domain Met 1 His 239 of human B7 2 NP 008820 2 was fused with a polyhistidine tag at the C terminus
    Catalog Number:
    10699-H08H
    Price:
    None
    Category:
    recombinant protein
    Host:
    HEK293 Cells
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    Structured Review

    Sino Biological human cd86
    PD-L1 Binds CD80 in Cis , and Atezolizumab Disrupts this Interaction (A) Representative TIRF images of PD-L1 LUVs captured by PD-1 SLB, CD80 SLB, or <t>CD86</t> SLB; each LUV is registered as a green spot. Bar graph summarizes the fluorescence intensity (FI) of the LUV channel under indicated conditions, normalized to the intensity of the condition with PD-1 SLBs. Data are means ± SEM, n = 3. Scale bars, 5 μm. (B) A FRET assay showing PD-L1:CD80 cis -interaction on cell membranes. Cartoons on the left depict a HEK293T cell co-expressing PD-L1 (labeled with CS547, donor) and either CD80 or CD86 (labeled with SSAF647, acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell at the indicated channels. Further right are calculated FRET efficiency images (pseudo-color; the yellow to purple spectrum denotes strong to weak FRET) and the differential interference contrast (DIC) images. Rightmost are bar graphs summarizing the FRET efficiencies as mean ± SEM, n > 25 cells from 3 independent experiments. Scale bars, 10 μm. (C) Same as (B) except replacing PD-L1 with PD-L2. (D) On the left is a cartoon depicting an LUV FRET assay for probing PD-L1:CD80 cis -interaction and atezolizumab (Atezo) effects. SC505 (donor) labeled SNAP-PD-L1-His was pre-bound to LUVs via DGS-NTA-Ni. Subsequently added TMR (acceptor) labeled SNAP-CD80-His bound to the LUVs and interacted with PD-L1 in cis , causing FRET and SC505 quenching (black trace). On the right are time courses of normalized SC505 fluorescence under the indicated conditions. Color coding is as follows: blue, same as black except plus atezolizumab; magenta, same as black except using TMR*CD80 lacking a His tag; orange, same as black except replacing PD-L1 with PD-L2; gray, same as black except presenting TMR*CD80 in trans . Data are representative of 3 independent replicates. Unpaired two-tailed Student’s t test: *p
    A DNA sequence encoding the extracellular domain Met 1 His 239 of human B7 2 NP 008820 2 was fused with a polyhistidine tag at the C terminus
    https://www.bioz.com/result/human cd86/product/Sino Biological
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human cd86 - by Bioz Stars, 2021-04
    92/100 stars

    Images

    1) Product Images from "PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways"

    Article Title: PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways

    Journal: Immunity

    doi: 10.1016/j.immuni.2019.11.003

    PD-L1 Binds CD80 in Cis , and Atezolizumab Disrupts this Interaction (A) Representative TIRF images of PD-L1 LUVs captured by PD-1 SLB, CD80 SLB, or CD86 SLB; each LUV is registered as a green spot. Bar graph summarizes the fluorescence intensity (FI) of the LUV channel under indicated conditions, normalized to the intensity of the condition with PD-1 SLBs. Data are means ± SEM, n = 3. Scale bars, 5 μm. (B) A FRET assay showing PD-L1:CD80 cis -interaction on cell membranes. Cartoons on the left depict a HEK293T cell co-expressing PD-L1 (labeled with CS547, donor) and either CD80 or CD86 (labeled with SSAF647, acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell at the indicated channels. Further right are calculated FRET efficiency images (pseudo-color; the yellow to purple spectrum denotes strong to weak FRET) and the differential interference contrast (DIC) images. Rightmost are bar graphs summarizing the FRET efficiencies as mean ± SEM, n > 25 cells from 3 independent experiments. Scale bars, 10 μm. (C) Same as (B) except replacing PD-L1 with PD-L2. (D) On the left is a cartoon depicting an LUV FRET assay for probing PD-L1:CD80 cis -interaction and atezolizumab (Atezo) effects. SC505 (donor) labeled SNAP-PD-L1-His was pre-bound to LUVs via DGS-NTA-Ni. Subsequently added TMR (acceptor) labeled SNAP-CD80-His bound to the LUVs and interacted with PD-L1 in cis , causing FRET and SC505 quenching (black trace). On the right are time courses of normalized SC505 fluorescence under the indicated conditions. Color coding is as follows: blue, same as black except plus atezolizumab; magenta, same as black except using TMR*CD80 lacking a His tag; orange, same as black except replacing PD-L1 with PD-L2; gray, same as black except presenting TMR*CD80 in trans . Data are representative of 3 independent replicates. Unpaired two-tailed Student’s t test: *p
    Figure Legend Snippet: PD-L1 Binds CD80 in Cis , and Atezolizumab Disrupts this Interaction (A) Representative TIRF images of PD-L1 LUVs captured by PD-1 SLB, CD80 SLB, or CD86 SLB; each LUV is registered as a green spot. Bar graph summarizes the fluorescence intensity (FI) of the LUV channel under indicated conditions, normalized to the intensity of the condition with PD-1 SLBs. Data are means ± SEM, n = 3. Scale bars, 5 μm. (B) A FRET assay showing PD-L1:CD80 cis -interaction on cell membranes. Cartoons on the left depict a HEK293T cell co-expressing PD-L1 (labeled with CS547, donor) and either CD80 or CD86 (labeled with SSAF647, acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell at the indicated channels. Further right are calculated FRET efficiency images (pseudo-color; the yellow to purple spectrum denotes strong to weak FRET) and the differential interference contrast (DIC) images. Rightmost are bar graphs summarizing the FRET efficiencies as mean ± SEM, n > 25 cells from 3 independent experiments. Scale bars, 10 μm. (C) Same as (B) except replacing PD-L1 with PD-L2. (D) On the left is a cartoon depicting an LUV FRET assay for probing PD-L1:CD80 cis -interaction and atezolizumab (Atezo) effects. SC505 (donor) labeled SNAP-PD-L1-His was pre-bound to LUVs via DGS-NTA-Ni. Subsequently added TMR (acceptor) labeled SNAP-CD80-His bound to the LUVs and interacted with PD-L1 in cis , causing FRET and SC505 quenching (black trace). On the right are time courses of normalized SC505 fluorescence under the indicated conditions. Color coding is as follows: blue, same as black except plus atezolizumab; magenta, same as black except using TMR*CD80 lacking a His tag; orange, same as black except replacing PD-L1 with PD-L2; gray, same as black except presenting TMR*CD80 in trans . Data are representative of 3 independent replicates. Unpaired two-tailed Student’s t test: *p

    Techniques Used: Fluorescence, Expressing, Labeling, Two Tailed Test

    Cis -PD-L1 Inhibits CD80:CTLA-4 Interaction through Disrupting CD80 Homodimers (A) Representative flow-cytometry histograms of CTLA-4-huFc staining of the indicated types of Raji cells. Bound CTLA-4-huFc was labeled by AF647 anti-human IgG Fc, the MFI of which was plotted against (CTLA-4-huFc). Shown in gray are Raji (CD80 + CD86 − ) cells stained by isolated huFc domain. Means ± SEM, n ≥ 3. (B) Representative flow-cytometry histograms of CTLA-4-moFc staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab (Atezo) (20 μg/mL). Bound moFc was labeled by AF647 anti-mouse IgG Fc, the MFI of which was plotted against (CTLA-4-moFc). Shown in gray are atezolizumab-treated Raji (CD80 + CD86 − PD-L1-mCherry + ) cells stained by isolated moFc domain. Means ± SEM, n ≥ 3. (C) Representative flow-cytometry histograms of CTLA-4-GCN4*SC647 staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab and of Raji (CD80 − CD86 − ) cells with atezolizumab. MFI of SC647 was plotted against the input concentration (means ± SEM, n ≥ 3). (D) At the top are flow-cytometry histograms showing both PD-L1 and CD80 amounts on a population of Raji (CD80 wd CD86 − PD-L1-mCherry + ) cells with tight PD-L1 expression and a wide range of CD80 expression. The cells were stained with either phycoerythrin (PE) anti-CD80, PE anti-PD-L1, or PE isotype, and the 3 histograms overlaid. On the bottom is a flow-cytometry dot plot showing CTLA-4-GCN4*SC647 staining of Raji (CD80 wd CD86 − PD-L1-mCherry + ) cells with or without atezolizumab. Gray dots correspond to control signals of unstained cells. CD80 + cells were gated by the vertical dash line, determined by the mGFP signal of parental Raji (CD80 − CD86 − ) cells. (E) A FRET assay probing CD80:CD80 homodimerization on cell membranes. In the first row, the leftmost cartoon depicts a HEK293T cell expressing SNAP-CD80, with a subpopulation labeled with SS549 (donor) and the rest labeled with SSAF647 (acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell. Further on the right is the calculated pseudo-color FRET efficiency image (yellow to purple spectrum denotes strong to weak FRET) and the DIC image. The second and third rows are the same as the first row except replacing SNAP-CD80 with SNAP-CD80 (I92R) or with SNAP-CD86. The fourth row is the same as the first row except with co-expressed unlabeled PD-L1. The fifth row is the same as fourth row except in the presence of atezolizumab. The bar graph summarizes the FRET efficiencies as mean ± SEM, n > 22 cells from 3 independent experiments. Scale bars, 10 μm. (F) An LUV FRET assay for probing CD80:CD80 homodimerization and PD-L1 effects. Shown is a representative time course of normalized FI of LUV-bound SC505*CD80-His, challenged by TMR*CD80-His and then by indicated concentrations of unlabeled PD-L1-His, with or without atezolizumab (Atezo) (20 μg/mL). (G) An LUV FRET assay showing that a single point mutation in CD80 disrupts both CD80:CD80 homodimerization and PD-L1:CD80 heterodimerization. Each indicated SC505 (energy donor)-labeled protein was pre-coupled to DGS-NTA-Ni containing LUVs through its His-tag, and challenged with TMR (energy acceptor)-labeled proteins as indicated. Shown are representative time courses of 3 independent replicates. (H) Representative TIRF images of Raji (CD80-mGFP + CD86 − PD-L1-SNAP + . Unpaired two-tailed Student’s for genotypes of cells related to this figure.
    Figure Legend Snippet: Cis -PD-L1 Inhibits CD80:CTLA-4 Interaction through Disrupting CD80 Homodimers (A) Representative flow-cytometry histograms of CTLA-4-huFc staining of the indicated types of Raji cells. Bound CTLA-4-huFc was labeled by AF647 anti-human IgG Fc, the MFI of which was plotted against (CTLA-4-huFc). Shown in gray are Raji (CD80 + CD86 − ) cells stained by isolated huFc domain. Means ± SEM, n ≥ 3. (B) Representative flow-cytometry histograms of CTLA-4-moFc staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab (Atezo) (20 μg/mL). Bound moFc was labeled by AF647 anti-mouse IgG Fc, the MFI of which was plotted against (CTLA-4-moFc). Shown in gray are atezolizumab-treated Raji (CD80 + CD86 − PD-L1-mCherry + ) cells stained by isolated moFc domain. Means ± SEM, n ≥ 3. (C) Representative flow-cytometry histograms of CTLA-4-GCN4*SC647 staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab and of Raji (CD80 − CD86 − ) cells with atezolizumab. MFI of SC647 was plotted against the input concentration (means ± SEM, n ≥ 3). (D) At the top are flow-cytometry histograms showing both PD-L1 and CD80 amounts on a population of Raji (CD80 wd CD86 − PD-L1-mCherry + ) cells with tight PD-L1 expression and a wide range of CD80 expression. The cells were stained with either phycoerythrin (PE) anti-CD80, PE anti-PD-L1, or PE isotype, and the 3 histograms overlaid. On the bottom is a flow-cytometry dot plot showing CTLA-4-GCN4*SC647 staining of Raji (CD80 wd CD86 − PD-L1-mCherry + ) cells with or without atezolizumab. Gray dots correspond to control signals of unstained cells. CD80 + cells were gated by the vertical dash line, determined by the mGFP signal of parental Raji (CD80 − CD86 − ) cells. (E) A FRET assay probing CD80:CD80 homodimerization on cell membranes. In the first row, the leftmost cartoon depicts a HEK293T cell expressing SNAP-CD80, with a subpopulation labeled with SS549 (donor) and the rest labeled with SSAF647 (acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell. Further on the right is the calculated pseudo-color FRET efficiency image (yellow to purple spectrum denotes strong to weak FRET) and the DIC image. The second and third rows are the same as the first row except replacing SNAP-CD80 with SNAP-CD80 (I92R) or with SNAP-CD86. The fourth row is the same as the first row except with co-expressed unlabeled PD-L1. The fifth row is the same as fourth row except in the presence of atezolizumab. The bar graph summarizes the FRET efficiencies as mean ± SEM, n > 22 cells from 3 independent experiments. Scale bars, 10 μm. (F) An LUV FRET assay for probing CD80:CD80 homodimerization and PD-L1 effects. Shown is a representative time course of normalized FI of LUV-bound SC505*CD80-His, challenged by TMR*CD80-His and then by indicated concentrations of unlabeled PD-L1-His, with or without atezolizumab (Atezo) (20 μg/mL). (G) An LUV FRET assay showing that a single point mutation in CD80 disrupts both CD80:CD80 homodimerization and PD-L1:CD80 heterodimerization. Each indicated SC505 (energy donor)-labeled protein was pre-coupled to DGS-NTA-Ni containing LUVs through its His-tag, and challenged with TMR (energy acceptor)-labeled proteins as indicated. Shown are representative time courses of 3 independent replicates. (H) Representative TIRF images of Raji (CD80-mGFP + CD86 − PD-L1-SNAP + . Unpaired two-tailed Student’s for genotypes of cells related to this figure.

    Techniques Used: Flow Cytometry, Cytometry, Staining, Labeling, Isolation, Concentration Assay, Expressing, Mutagenesis, Two Tailed Test

    Cis -PD-L1 Protects CD80 from CTLA-4 Mediated Trans -Endocytosis (A) A Jurkat-Raji co-culture assay analyzing how PD-L1 interferes with CTLA-4-mediated CD80 depletion. Cartoons on the left depict the co-cultured cells. On the immediate right are representative flow-cytometry histograms of CD80 expression (anti-CD80 allophycocyanin) on Raji cells before (0 h) and after co-culture (0.5 h). Further on the right are representative confocal images for the Jurkat-Raji conjugate (scale bars, 10 μm). Rightmost is a bar graph showing CD80 MFI of Raji at 0.5 h, normalized to CD80 MFI at 0 h (mean ± SEM, n = 4). (B) An independent Jurkat-Raji conjugation assay examining how anti-PD-L1 and anti-CTLA-4 affect CD80 amounts. Experiments were conducted as in (A) except pretreating the indicated cell type with atezolizumab (Atezo) or ipilimumab (Ipi), as depicted in the cartoons. On the immediate right are representative flow-cytometry histograms of CD80 expression and confocal images for the Jurkat-Raji conjugate (scale bars, 10 μm). Rightmost is a bar graph showing CD80 MFI of Raji at 0.5 h, normalized to CD80 MFI at 0 h (mean ± SEM, n = 5). (C) Representative flow-cytometry histograms of CD80 and CD86 surface expressions on mouse splenic DCs co-cultured with either Tconv or Treg cells with or without the indicated checkpoint inhibitors for 16 h. Bar graphs summarize the CD80 and CD86 MFI (mean ± SEM, n = 3). . Unpaired two-tailed Student’s for genotypes of cells related to this figure.
    Figure Legend Snippet: Cis -PD-L1 Protects CD80 from CTLA-4 Mediated Trans -Endocytosis (A) A Jurkat-Raji co-culture assay analyzing how PD-L1 interferes with CTLA-4-mediated CD80 depletion. Cartoons on the left depict the co-cultured cells. On the immediate right are representative flow-cytometry histograms of CD80 expression (anti-CD80 allophycocyanin) on Raji cells before (0 h) and after co-culture (0.5 h). Further on the right are representative confocal images for the Jurkat-Raji conjugate (scale bars, 10 μm). Rightmost is a bar graph showing CD80 MFI of Raji at 0.5 h, normalized to CD80 MFI at 0 h (mean ± SEM, n = 4). (B) An independent Jurkat-Raji conjugation assay examining how anti-PD-L1 and anti-CTLA-4 affect CD80 amounts. Experiments were conducted as in (A) except pretreating the indicated cell type with atezolizumab (Atezo) or ipilimumab (Ipi), as depicted in the cartoons. On the immediate right are representative flow-cytometry histograms of CD80 expression and confocal images for the Jurkat-Raji conjugate (scale bars, 10 μm). Rightmost is a bar graph showing CD80 MFI of Raji at 0.5 h, normalized to CD80 MFI at 0 h (mean ± SEM, n = 5). (C) Representative flow-cytometry histograms of CD80 and CD86 surface expressions on mouse splenic DCs co-cultured with either Tconv or Treg cells with or without the indicated checkpoint inhibitors for 16 h. Bar graphs summarize the CD80 and CD86 MFI (mean ± SEM, n = 3). . Unpaired two-tailed Student’s for genotypes of cells related to this figure.

    Techniques Used: Co-culture Assay, Cell Culture, Flow Cytometry, Cytometry, Expressing, Co-Culture Assay, Conjugation Assay, Two Tailed Test

    Cis -PD-L1 Does Not Affect CD80:CD28 Interaction (A) Representative flow-cytometry histograms of CD28-huFc staining of the indicated types of Raji cells. Bound CD28-huFc was labeled by AF647 anti-human IgG Fc, the MFI of which was plotted against (CD28-huFc. Shown in gray are Raji (CD80 + CD86 − ) cells stained by isolated huFc domain. Means ± SEM, n = 3. (B) Representative flow-cytometry histograms of CD28-moFc staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab (Atezo) (20 μg/mL). Bound moFc was labeled by AF647 anti-mouse IgG Fc, the MFI of which was plotted against (CD28-moFc). Shown in gray are atezolizumab-treated Raji (CD80 + CD86 − PD-L1-mCherry + ) cells stained by isolated moFc domain. Means ± SEM, n = 3. (C) A T-cell-SLB assay showing cis -PD-L1 effects on CD80-induced CD28 microclusters. On the left is a cartoon for a CD28-mGFP transduced OT-1 cell interacting with an SLB containing pMHC, ICAM (not shown), and CD80. On the immediate right are TIRF images of CD28-mGFP (rendered in green) and TCR stained by AF647-labeled H57–597 TCR-β antibody(rendered in magenta) 30 s after cells contacted the pMHC- and ICAM-containing SLB supplemented with the indicated ligands. The bar graph shows the clustering indices of CD28 and TCR under each condition (means ± SEM of ≥ 18 cells from 3 independent experiments). Scale bars, 5 μm. (D) A T-cell-APC conjugate assay probing how cis -PD-L1 affects CD80:CD28 interaction. The leftmost cartoons depict a Jurkat (CD28-mCherry + ) cell forming a conjugate with a Raji (CD80-mGFP + CD86 + ) cell, a Raji (CD80-mGFP + CD86 + CLIP-PD-L1 + ) cell, or a Raji (CD86 + CLIP-PD-L1 + ) cell. On the immediate right are confocal images of a cell conjugate acquired 2 min after Jurkat-Raji contact. The bar graph summarizes the synaptic enrichment indices of the 3 conditions with CD28 rendered in magenta, CD80 in green, and PD-L1 in blue (means ± SEM, n = 30 conjugates from 3 independent experiments). Scale bars, 10 μm. (E) At the top is a representative IB showing CD28:p85 co-IP from the lysates of the indicated co-cultures, with the times of lysis denoted. On the bottom is a quantification bar graph (means ± SEM, n = 3). . Unpaired two-tailed Student’s for genotypes of cells related to this figure.
    Figure Legend Snippet: Cis -PD-L1 Does Not Affect CD80:CD28 Interaction (A) Representative flow-cytometry histograms of CD28-huFc staining of the indicated types of Raji cells. Bound CD28-huFc was labeled by AF647 anti-human IgG Fc, the MFI of which was plotted against (CD28-huFc. Shown in gray are Raji (CD80 + CD86 − ) cells stained by isolated huFc domain. Means ± SEM, n = 3. (B) Representative flow-cytometry histograms of CD28-moFc staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab (Atezo) (20 μg/mL). Bound moFc was labeled by AF647 anti-mouse IgG Fc, the MFI of which was plotted against (CD28-moFc). Shown in gray are atezolizumab-treated Raji (CD80 + CD86 − PD-L1-mCherry + ) cells stained by isolated moFc domain. Means ± SEM, n = 3. (C) A T-cell-SLB assay showing cis -PD-L1 effects on CD80-induced CD28 microclusters. On the left is a cartoon for a CD28-mGFP transduced OT-1 cell interacting with an SLB containing pMHC, ICAM (not shown), and CD80. On the immediate right are TIRF images of CD28-mGFP (rendered in green) and TCR stained by AF647-labeled H57–597 TCR-β antibody(rendered in magenta) 30 s after cells contacted the pMHC- and ICAM-containing SLB supplemented with the indicated ligands. The bar graph shows the clustering indices of CD28 and TCR under each condition (means ± SEM of ≥ 18 cells from 3 independent experiments). Scale bars, 5 μm. (D) A T-cell-APC conjugate assay probing how cis -PD-L1 affects CD80:CD28 interaction. The leftmost cartoons depict a Jurkat (CD28-mCherry + ) cell forming a conjugate with a Raji (CD80-mGFP + CD86 + ) cell, a Raji (CD80-mGFP + CD86 + CLIP-PD-L1 + ) cell, or a Raji (CD86 + CLIP-PD-L1 + ) cell. On the immediate right are confocal images of a cell conjugate acquired 2 min after Jurkat-Raji contact. The bar graph summarizes the synaptic enrichment indices of the 3 conditions with CD28 rendered in magenta, CD80 in green, and PD-L1 in blue (means ± SEM, n = 30 conjugates from 3 independent experiments). Scale bars, 10 μm. (E) At the top is a representative IB showing CD28:p85 co-IP from the lysates of the indicated co-cultures, with the times of lysis denoted. On the bottom is a quantification bar graph (means ± SEM, n = 3). . Unpaired two-tailed Student’s for genotypes of cells related to this figure.

    Techniques Used: Flow Cytometry, Cytometry, Staining, Labeling, Isolation, Cross-linking Immunoprecipitation, Co-Immunoprecipitation Assay, Lysis, Two Tailed Test

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    Sino Biological human cd86
    PD-L1 Binds CD80 in Cis , and Atezolizumab Disrupts this Interaction (A) Representative TIRF images of PD-L1 LUVs captured by PD-1 SLB, CD80 SLB, or <t>CD86</t> SLB; each LUV is registered as a green spot. Bar graph summarizes the fluorescence intensity (FI) of the LUV channel under indicated conditions, normalized to the intensity of the condition with PD-1 SLBs. Data are means ± SEM, n = 3. Scale bars, 5 μm. (B) A FRET assay showing PD-L1:CD80 cis -interaction on cell membranes. Cartoons on the left depict a HEK293T cell co-expressing PD-L1 (labeled with CS547, donor) and either CD80 or CD86 (labeled with SSAF647, acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell at the indicated channels. Further right are calculated FRET efficiency images (pseudo-color; the yellow to purple spectrum denotes strong to weak FRET) and the differential interference contrast (DIC) images. Rightmost are bar graphs summarizing the FRET efficiencies as mean ± SEM, n > 25 cells from 3 independent experiments. Scale bars, 10 μm. (C) Same as (B) except replacing PD-L1 with PD-L2. (D) On the left is a cartoon depicting an LUV FRET assay for probing PD-L1:CD80 cis -interaction and atezolizumab (Atezo) effects. SC505 (donor) labeled SNAP-PD-L1-His was pre-bound to LUVs via DGS-NTA-Ni. Subsequently added TMR (acceptor) labeled SNAP-CD80-His bound to the LUVs and interacted with PD-L1 in cis , causing FRET and SC505 quenching (black trace). On the right are time courses of normalized SC505 fluorescence under the indicated conditions. Color coding is as follows: blue, same as black except plus atezolizumab; magenta, same as black except using TMR*CD80 lacking a His tag; orange, same as black except replacing PD-L1 with PD-L2; gray, same as black except presenting TMR*CD80 in trans . Data are representative of 3 independent replicates. Unpaired two-tailed Student’s t test: *p
    Human Cd86, supplied by Sino Biological, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human cd86/product/Sino Biological
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    human cd86 - by Bioz Stars, 2021-04
    92/100 stars
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    PD-L1 Binds CD80 in Cis , and Atezolizumab Disrupts this Interaction (A) Representative TIRF images of PD-L1 LUVs captured by PD-1 SLB, CD80 SLB, or CD86 SLB; each LUV is registered as a green spot. Bar graph summarizes the fluorescence intensity (FI) of the LUV channel under indicated conditions, normalized to the intensity of the condition with PD-1 SLBs. Data are means ± SEM, n = 3. Scale bars, 5 μm. (B) A FRET assay showing PD-L1:CD80 cis -interaction on cell membranes. Cartoons on the left depict a HEK293T cell co-expressing PD-L1 (labeled with CS547, donor) and either CD80 or CD86 (labeled with SSAF647, acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell at the indicated channels. Further right are calculated FRET efficiency images (pseudo-color; the yellow to purple spectrum denotes strong to weak FRET) and the differential interference contrast (DIC) images. Rightmost are bar graphs summarizing the FRET efficiencies as mean ± SEM, n > 25 cells from 3 independent experiments. Scale bars, 10 μm. (C) Same as (B) except replacing PD-L1 with PD-L2. (D) On the left is a cartoon depicting an LUV FRET assay for probing PD-L1:CD80 cis -interaction and atezolizumab (Atezo) effects. SC505 (donor) labeled SNAP-PD-L1-His was pre-bound to LUVs via DGS-NTA-Ni. Subsequently added TMR (acceptor) labeled SNAP-CD80-His bound to the LUVs and interacted with PD-L1 in cis , causing FRET and SC505 quenching (black trace). On the right are time courses of normalized SC505 fluorescence under the indicated conditions. Color coding is as follows: blue, same as black except plus atezolizumab; magenta, same as black except using TMR*CD80 lacking a His tag; orange, same as black except replacing PD-L1 with PD-L2; gray, same as black except presenting TMR*CD80 in trans . Data are representative of 3 independent replicates. Unpaired two-tailed Student’s t test: *p

    Journal: Immunity

    Article Title: PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways

    doi: 10.1016/j.immuni.2019.11.003

    Figure Lengend Snippet: PD-L1 Binds CD80 in Cis , and Atezolizumab Disrupts this Interaction (A) Representative TIRF images of PD-L1 LUVs captured by PD-1 SLB, CD80 SLB, or CD86 SLB; each LUV is registered as a green spot. Bar graph summarizes the fluorescence intensity (FI) of the LUV channel under indicated conditions, normalized to the intensity of the condition with PD-1 SLBs. Data are means ± SEM, n = 3. Scale bars, 5 μm. (B) A FRET assay showing PD-L1:CD80 cis -interaction on cell membranes. Cartoons on the left depict a HEK293T cell co-expressing PD-L1 (labeled with CS547, donor) and either CD80 or CD86 (labeled with SSAF647, acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell at the indicated channels. Further right are calculated FRET efficiency images (pseudo-color; the yellow to purple spectrum denotes strong to weak FRET) and the differential interference contrast (DIC) images. Rightmost are bar graphs summarizing the FRET efficiencies as mean ± SEM, n > 25 cells from 3 independent experiments. Scale bars, 10 μm. (C) Same as (B) except replacing PD-L1 with PD-L2. (D) On the left is a cartoon depicting an LUV FRET assay for probing PD-L1:CD80 cis -interaction and atezolizumab (Atezo) effects. SC505 (donor) labeled SNAP-PD-L1-His was pre-bound to LUVs via DGS-NTA-Ni. Subsequently added TMR (acceptor) labeled SNAP-CD80-His bound to the LUVs and interacted with PD-L1 in cis , causing FRET and SC505 quenching (black trace). On the right are time courses of normalized SC505 fluorescence under the indicated conditions. Color coding is as follows: blue, same as black except plus atezolizumab; magenta, same as black except using TMR*CD80 lacking a His tag; orange, same as black except replacing PD-L1 with PD-L2; gray, same as black except presenting TMR*CD80 in trans . Data are representative of 3 independent replicates. Unpaired two-tailed Student’s t test: *p

    Article Snippet: The SLBs were then overlaid with 200 μL of either 3 nM human PD-1-His (Sino Biological, 10377-H08H), 3 nM human CD80–His (Sino Biological, 10698-H08H), or 3 nM human CD86–His (Sino Biological, 10699-H08H).

    Techniques: Fluorescence, Expressing, Labeling, Two Tailed Test

    Cis -PD-L1 Inhibits CD80:CTLA-4 Interaction through Disrupting CD80 Homodimers (A) Representative flow-cytometry histograms of CTLA-4-huFc staining of the indicated types of Raji cells. Bound CTLA-4-huFc was labeled by AF647 anti-human IgG Fc, the MFI of which was plotted against (CTLA-4-huFc). Shown in gray are Raji (CD80 + CD86 − ) cells stained by isolated huFc domain. Means ± SEM, n ≥ 3. (B) Representative flow-cytometry histograms of CTLA-4-moFc staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab (Atezo) (20 μg/mL). Bound moFc was labeled by AF647 anti-mouse IgG Fc, the MFI of which was plotted against (CTLA-4-moFc). Shown in gray are atezolizumab-treated Raji (CD80 + CD86 − PD-L1-mCherry + ) cells stained by isolated moFc domain. Means ± SEM, n ≥ 3. (C) Representative flow-cytometry histograms of CTLA-4-GCN4*SC647 staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab and of Raji (CD80 − CD86 − ) cells with atezolizumab. MFI of SC647 was plotted against the input concentration (means ± SEM, n ≥ 3). (D) At the top are flow-cytometry histograms showing both PD-L1 and CD80 amounts on a population of Raji (CD80 wd CD86 − PD-L1-mCherry + ) cells with tight PD-L1 expression and a wide range of CD80 expression. The cells were stained with either phycoerythrin (PE) anti-CD80, PE anti-PD-L1, or PE isotype, and the 3 histograms overlaid. On the bottom is a flow-cytometry dot plot showing CTLA-4-GCN4*SC647 staining of Raji (CD80 wd CD86 − PD-L1-mCherry + ) cells with or without atezolizumab. Gray dots correspond to control signals of unstained cells. CD80 + cells were gated by the vertical dash line, determined by the mGFP signal of parental Raji (CD80 − CD86 − ) cells. (E) A FRET assay probing CD80:CD80 homodimerization on cell membranes. In the first row, the leftmost cartoon depicts a HEK293T cell expressing SNAP-CD80, with a subpopulation labeled with SS549 (donor) and the rest labeled with SSAF647 (acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell. Further on the right is the calculated pseudo-color FRET efficiency image (yellow to purple spectrum denotes strong to weak FRET) and the DIC image. The second and third rows are the same as the first row except replacing SNAP-CD80 with SNAP-CD80 (I92R) or with SNAP-CD86. The fourth row is the same as the first row except with co-expressed unlabeled PD-L1. The fifth row is the same as fourth row except in the presence of atezolizumab. The bar graph summarizes the FRET efficiencies as mean ± SEM, n > 22 cells from 3 independent experiments. Scale bars, 10 μm. (F) An LUV FRET assay for probing CD80:CD80 homodimerization and PD-L1 effects. Shown is a representative time course of normalized FI of LUV-bound SC505*CD80-His, challenged by TMR*CD80-His and then by indicated concentrations of unlabeled PD-L1-His, with or without atezolizumab (Atezo) (20 μg/mL). (G) An LUV FRET assay showing that a single point mutation in CD80 disrupts both CD80:CD80 homodimerization and PD-L1:CD80 heterodimerization. Each indicated SC505 (energy donor)-labeled protein was pre-coupled to DGS-NTA-Ni containing LUVs through its His-tag, and challenged with TMR (energy acceptor)-labeled proteins as indicated. Shown are representative time courses of 3 independent replicates. (H) Representative TIRF images of Raji (CD80-mGFP + CD86 − PD-L1-SNAP + . Unpaired two-tailed Student’s for genotypes of cells related to this figure.

    Journal: Immunity

    Article Title: PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways

    doi: 10.1016/j.immuni.2019.11.003

    Figure Lengend Snippet: Cis -PD-L1 Inhibits CD80:CTLA-4 Interaction through Disrupting CD80 Homodimers (A) Representative flow-cytometry histograms of CTLA-4-huFc staining of the indicated types of Raji cells. Bound CTLA-4-huFc was labeled by AF647 anti-human IgG Fc, the MFI of which was plotted against (CTLA-4-huFc). Shown in gray are Raji (CD80 + CD86 − ) cells stained by isolated huFc domain. Means ± SEM, n ≥ 3. (B) Representative flow-cytometry histograms of CTLA-4-moFc staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab (Atezo) (20 μg/mL). Bound moFc was labeled by AF647 anti-mouse IgG Fc, the MFI of which was plotted against (CTLA-4-moFc). Shown in gray are atezolizumab-treated Raji (CD80 + CD86 − PD-L1-mCherry + ) cells stained by isolated moFc domain. Means ± SEM, n ≥ 3. (C) Representative flow-cytometry histograms of CTLA-4-GCN4*SC647 staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab and of Raji (CD80 − CD86 − ) cells with atezolizumab. MFI of SC647 was plotted against the input concentration (means ± SEM, n ≥ 3). (D) At the top are flow-cytometry histograms showing both PD-L1 and CD80 amounts on a population of Raji (CD80 wd CD86 − PD-L1-mCherry + ) cells with tight PD-L1 expression and a wide range of CD80 expression. The cells were stained with either phycoerythrin (PE) anti-CD80, PE anti-PD-L1, or PE isotype, and the 3 histograms overlaid. On the bottom is a flow-cytometry dot plot showing CTLA-4-GCN4*SC647 staining of Raji (CD80 wd CD86 − PD-L1-mCherry + ) cells with or without atezolizumab. Gray dots correspond to control signals of unstained cells. CD80 + cells were gated by the vertical dash line, determined by the mGFP signal of parental Raji (CD80 − CD86 − ) cells. (E) A FRET assay probing CD80:CD80 homodimerization on cell membranes. In the first row, the leftmost cartoon depicts a HEK293T cell expressing SNAP-CD80, with a subpopulation labeled with SS549 (donor) and the rest labeled with SSAF647 (acceptor). On the immediate right are pre- and post-bleaching confocal images of a representative cell. Further on the right is the calculated pseudo-color FRET efficiency image (yellow to purple spectrum denotes strong to weak FRET) and the DIC image. The second and third rows are the same as the first row except replacing SNAP-CD80 with SNAP-CD80 (I92R) or with SNAP-CD86. The fourth row is the same as the first row except with co-expressed unlabeled PD-L1. The fifth row is the same as fourth row except in the presence of atezolizumab. The bar graph summarizes the FRET efficiencies as mean ± SEM, n > 22 cells from 3 independent experiments. Scale bars, 10 μm. (F) An LUV FRET assay for probing CD80:CD80 homodimerization and PD-L1 effects. Shown is a representative time course of normalized FI of LUV-bound SC505*CD80-His, challenged by TMR*CD80-His and then by indicated concentrations of unlabeled PD-L1-His, with or without atezolizumab (Atezo) (20 μg/mL). (G) An LUV FRET assay showing that a single point mutation in CD80 disrupts both CD80:CD80 homodimerization and PD-L1:CD80 heterodimerization. Each indicated SC505 (energy donor)-labeled protein was pre-coupled to DGS-NTA-Ni containing LUVs through its His-tag, and challenged with TMR (energy acceptor)-labeled proteins as indicated. Shown are representative time courses of 3 independent replicates. (H) Representative TIRF images of Raji (CD80-mGFP + CD86 − PD-L1-SNAP + . Unpaired two-tailed Student’s for genotypes of cells related to this figure.

    Article Snippet: The SLBs were then overlaid with 200 μL of either 3 nM human PD-1-His (Sino Biological, 10377-H08H), 3 nM human CD80–His (Sino Biological, 10698-H08H), or 3 nM human CD86–His (Sino Biological, 10699-H08H).

    Techniques: Flow Cytometry, Cytometry, Staining, Labeling, Isolation, Concentration Assay, Expressing, Mutagenesis, Two Tailed Test

    Cis -PD-L1 Protects CD80 from CTLA-4 Mediated Trans -Endocytosis (A) A Jurkat-Raji co-culture assay analyzing how PD-L1 interferes with CTLA-4-mediated CD80 depletion. Cartoons on the left depict the co-cultured cells. On the immediate right are representative flow-cytometry histograms of CD80 expression (anti-CD80 allophycocyanin) on Raji cells before (0 h) and after co-culture (0.5 h). Further on the right are representative confocal images for the Jurkat-Raji conjugate (scale bars, 10 μm). Rightmost is a bar graph showing CD80 MFI of Raji at 0.5 h, normalized to CD80 MFI at 0 h (mean ± SEM, n = 4). (B) An independent Jurkat-Raji conjugation assay examining how anti-PD-L1 and anti-CTLA-4 affect CD80 amounts. Experiments were conducted as in (A) except pretreating the indicated cell type with atezolizumab (Atezo) or ipilimumab (Ipi), as depicted in the cartoons. On the immediate right are representative flow-cytometry histograms of CD80 expression and confocal images for the Jurkat-Raji conjugate (scale bars, 10 μm). Rightmost is a bar graph showing CD80 MFI of Raji at 0.5 h, normalized to CD80 MFI at 0 h (mean ± SEM, n = 5). (C) Representative flow-cytometry histograms of CD80 and CD86 surface expressions on mouse splenic DCs co-cultured with either Tconv or Treg cells with or without the indicated checkpoint inhibitors for 16 h. Bar graphs summarize the CD80 and CD86 MFI (mean ± SEM, n = 3). . Unpaired two-tailed Student’s for genotypes of cells related to this figure.

    Journal: Immunity

    Article Title: PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways

    doi: 10.1016/j.immuni.2019.11.003

    Figure Lengend Snippet: Cis -PD-L1 Protects CD80 from CTLA-4 Mediated Trans -Endocytosis (A) A Jurkat-Raji co-culture assay analyzing how PD-L1 interferes with CTLA-4-mediated CD80 depletion. Cartoons on the left depict the co-cultured cells. On the immediate right are representative flow-cytometry histograms of CD80 expression (anti-CD80 allophycocyanin) on Raji cells before (0 h) and after co-culture (0.5 h). Further on the right are representative confocal images for the Jurkat-Raji conjugate (scale bars, 10 μm). Rightmost is a bar graph showing CD80 MFI of Raji at 0.5 h, normalized to CD80 MFI at 0 h (mean ± SEM, n = 4). (B) An independent Jurkat-Raji conjugation assay examining how anti-PD-L1 and anti-CTLA-4 affect CD80 amounts. Experiments were conducted as in (A) except pretreating the indicated cell type with atezolizumab (Atezo) or ipilimumab (Ipi), as depicted in the cartoons. On the immediate right are representative flow-cytometry histograms of CD80 expression and confocal images for the Jurkat-Raji conjugate (scale bars, 10 μm). Rightmost is a bar graph showing CD80 MFI of Raji at 0.5 h, normalized to CD80 MFI at 0 h (mean ± SEM, n = 5). (C) Representative flow-cytometry histograms of CD80 and CD86 surface expressions on mouse splenic DCs co-cultured with either Tconv or Treg cells with or without the indicated checkpoint inhibitors for 16 h. Bar graphs summarize the CD80 and CD86 MFI (mean ± SEM, n = 3). . Unpaired two-tailed Student’s for genotypes of cells related to this figure.

    Article Snippet: The SLBs were then overlaid with 200 μL of either 3 nM human PD-1-His (Sino Biological, 10377-H08H), 3 nM human CD80–His (Sino Biological, 10698-H08H), or 3 nM human CD86–His (Sino Biological, 10699-H08H).

    Techniques: Co-culture Assay, Cell Culture, Flow Cytometry, Cytometry, Expressing, Co-Culture Assay, Conjugation Assay, Two Tailed Test

    Cis -PD-L1 Does Not Affect CD80:CD28 Interaction (A) Representative flow-cytometry histograms of CD28-huFc staining of the indicated types of Raji cells. Bound CD28-huFc was labeled by AF647 anti-human IgG Fc, the MFI of which was plotted against (CD28-huFc. Shown in gray are Raji (CD80 + CD86 − ) cells stained by isolated huFc domain. Means ± SEM, n = 3. (B) Representative flow-cytometry histograms of CD28-moFc staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab (Atezo) (20 μg/mL). Bound moFc was labeled by AF647 anti-mouse IgG Fc, the MFI of which was plotted against (CD28-moFc). Shown in gray are atezolizumab-treated Raji (CD80 + CD86 − PD-L1-mCherry + ) cells stained by isolated moFc domain. Means ± SEM, n = 3. (C) A T-cell-SLB assay showing cis -PD-L1 effects on CD80-induced CD28 microclusters. On the left is a cartoon for a CD28-mGFP transduced OT-1 cell interacting with an SLB containing pMHC, ICAM (not shown), and CD80. On the immediate right are TIRF images of CD28-mGFP (rendered in green) and TCR stained by AF647-labeled H57–597 TCR-β antibody(rendered in magenta) 30 s after cells contacted the pMHC- and ICAM-containing SLB supplemented with the indicated ligands. The bar graph shows the clustering indices of CD28 and TCR under each condition (means ± SEM of ≥ 18 cells from 3 independent experiments). Scale bars, 5 μm. (D) A T-cell-APC conjugate assay probing how cis -PD-L1 affects CD80:CD28 interaction. The leftmost cartoons depict a Jurkat (CD28-mCherry + ) cell forming a conjugate with a Raji (CD80-mGFP + CD86 + ) cell, a Raji (CD80-mGFP + CD86 + CLIP-PD-L1 + ) cell, or a Raji (CD86 + CLIP-PD-L1 + ) cell. On the immediate right are confocal images of a cell conjugate acquired 2 min after Jurkat-Raji contact. The bar graph summarizes the synaptic enrichment indices of the 3 conditions with CD28 rendered in magenta, CD80 in green, and PD-L1 in blue (means ± SEM, n = 30 conjugates from 3 independent experiments). Scale bars, 10 μm. (E) At the top is a representative IB showing CD28:p85 co-IP from the lysates of the indicated co-cultures, with the times of lysis denoted. On the bottom is a quantification bar graph (means ± SEM, n = 3). . Unpaired two-tailed Student’s for genotypes of cells related to this figure.

    Journal: Immunity

    Article Title: PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways

    doi: 10.1016/j.immuni.2019.11.003

    Figure Lengend Snippet: Cis -PD-L1 Does Not Affect CD80:CD28 Interaction (A) Representative flow-cytometry histograms of CD28-huFc staining of the indicated types of Raji cells. Bound CD28-huFc was labeled by AF647 anti-human IgG Fc, the MFI of which was plotted against (CD28-huFc. Shown in gray are Raji (CD80 + CD86 − ) cells stained by isolated huFc domain. Means ± SEM, n = 3. (B) Representative flow-cytometry histograms of CD28-moFc staining of Raji (CD80 + CD86 − PD-L1-mCherry + ) cells with or without atezolizumab (Atezo) (20 μg/mL). Bound moFc was labeled by AF647 anti-mouse IgG Fc, the MFI of which was plotted against (CD28-moFc). Shown in gray are atezolizumab-treated Raji (CD80 + CD86 − PD-L1-mCherry + ) cells stained by isolated moFc domain. Means ± SEM, n = 3. (C) A T-cell-SLB assay showing cis -PD-L1 effects on CD80-induced CD28 microclusters. On the left is a cartoon for a CD28-mGFP transduced OT-1 cell interacting with an SLB containing pMHC, ICAM (not shown), and CD80. On the immediate right are TIRF images of CD28-mGFP (rendered in green) and TCR stained by AF647-labeled H57–597 TCR-β antibody(rendered in magenta) 30 s after cells contacted the pMHC- and ICAM-containing SLB supplemented with the indicated ligands. The bar graph shows the clustering indices of CD28 and TCR under each condition (means ± SEM of ≥ 18 cells from 3 independent experiments). Scale bars, 5 μm. (D) A T-cell-APC conjugate assay probing how cis -PD-L1 affects CD80:CD28 interaction. The leftmost cartoons depict a Jurkat (CD28-mCherry + ) cell forming a conjugate with a Raji (CD80-mGFP + CD86 + ) cell, a Raji (CD80-mGFP + CD86 + CLIP-PD-L1 + ) cell, or a Raji (CD86 + CLIP-PD-L1 + ) cell. On the immediate right are confocal images of a cell conjugate acquired 2 min after Jurkat-Raji contact. The bar graph summarizes the synaptic enrichment indices of the 3 conditions with CD28 rendered in magenta, CD80 in green, and PD-L1 in blue (means ± SEM, n = 30 conjugates from 3 independent experiments). Scale bars, 10 μm. (E) At the top is a representative IB showing CD28:p85 co-IP from the lysates of the indicated co-cultures, with the times of lysis denoted. On the bottom is a quantification bar graph (means ± SEM, n = 3). . Unpaired two-tailed Student’s for genotypes of cells related to this figure.

    Article Snippet: The SLBs were then overlaid with 200 μL of either 3 nM human PD-1-His (Sino Biological, 10377-H08H), 3 nM human CD80–His (Sino Biological, 10698-H08H), or 3 nM human CD86–His (Sino Biological, 10699-H08H).

    Techniques: Flow Cytometry, Cytometry, Staining, Labeling, Isolation, Cross-linking Immunoprecipitation, Co-Immunoprecipitation Assay, Lysis, Two Tailed Test