mouse pd l1  (Sino Biological)


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    Name:
    Mouse PD L1 B7 H1 CD274 Protein His Tag
    Description:
    A DNA sequence encoding the mouse CD274 NP 068693 1 extracellular domain Met 1 Thr 238 was fused with a polyhistidine tag at the C terminus
    Catalog Number:
    50010-M08H
    Price:
    None
    Category:
    recombinant protein
    Host:
    HEK293 Cells
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    Structured Review

    Sino Biological mouse pd l1
    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 <t>SNAP-PD-L1-His</t> 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 mouse CD274 NP 068693 1 extracellular domain Met 1 Thr 238 was fused with a polyhistidine tag at the C terminus
    https://www.bioz.com/result/mouse pd l1/product/Sino Biological
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse pd l1 - by Bioz Stars, 2021-07
    93/100 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

    2) Product Images from "Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers"

    Article Title: Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers

    Journal: Oncotarget

    doi: 10.18632/oncotarget.16708

    Selection of anti-mouse-PD-L1 specific Nbs ( A ) Amino acid sequence alignment of the purified Nbs C3, C7, E2 and E4. The Nb sequence includes three complementarity-determining regions (CDR 1, 2, 3; indicated in red) and four framework regions (FR1-4, indicated in black). FRs are relatively conserved but CDRs vary widely among Nbs. ( B ) Affinity/kinetics SPR study of purified Nbs interacting with immobilized His-tagged recombinant mouse PD-L1 protein. Sensorgrams of different concentrations of the Nbs are shown ( n  = 1). ( C ) Representative flow cytometry results, showing staining of unmodified HEK293T cells (grey line) or HEK293T cells lentivirally modified to express mouse PD-L1 (293T moPD-L1, red line) with mAbs specific for mouse PD-L1 or Nbs C3, C7, E4 and E2 ( n  = 3).
    Figure Legend Snippet: Selection of anti-mouse-PD-L1 specific Nbs ( A ) Amino acid sequence alignment of the purified Nbs C3, C7, E2 and E4. The Nb sequence includes three complementarity-determining regions (CDR 1, 2, 3; indicated in red) and four framework regions (FR1-4, indicated in black). FRs are relatively conserved but CDRs vary widely among Nbs. ( B ) Affinity/kinetics SPR study of purified Nbs interacting with immobilized His-tagged recombinant mouse PD-L1 protein. Sensorgrams of different concentrations of the Nbs are shown ( n = 1). ( C ) Representative flow cytometry results, showing staining of unmodified HEK293T cells (grey line) or HEK293T cells lentivirally modified to express mouse PD-L1 (293T moPD-L1, red line) with mAbs specific for mouse PD-L1 or Nbs C3, C7, E4 and E2 ( n = 3).

    Techniques Used: Selection, Sequencing, Purification, SPR Assay, Recombinant, Flow Cytometry, Staining, Modification

    3) 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

    4) Product Images from "Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers"

    Article Title: Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers

    Journal: Oncotarget

    doi: 10.18632/oncotarget.16708

    Selection of anti-mouse-PD-L1 specific Nbs ( A ) Amino acid sequence alignment of the purified Nbs C3, C7, E2 and E4. The Nb sequence includes three complementarity-determining regions (CDR 1, 2, 3; indicated in red) and four framework regions (FR1-4, indicated in black). FRs are relatively conserved but CDRs vary widely among Nbs. ( B ) Affinity/kinetics SPR study of purified Nbs interacting with immobilized His-tagged recombinant mouse PD-L1 protein. Sensorgrams of different concentrations of the Nbs are shown ( n  = 1). ( C ) Representative flow cytometry results, showing staining of unmodified HEK293T cells (grey line) or HEK293T cells lentivirally modified to express mouse PD-L1 (293T moPD-L1, red line) with mAbs specific for mouse PD-L1 or Nbs C3, C7, E4 and E2 ( n  = 3).
    Figure Legend Snippet: Selection of anti-mouse-PD-L1 specific Nbs ( A ) Amino acid sequence alignment of the purified Nbs C3, C7, E2 and E4. The Nb sequence includes three complementarity-determining regions (CDR 1, 2, 3; indicated in red) and four framework regions (FR1-4, indicated in black). FRs are relatively conserved but CDRs vary widely among Nbs. ( B ) Affinity/kinetics SPR study of purified Nbs interacting with immobilized His-tagged recombinant mouse PD-L1 protein. Sensorgrams of different concentrations of the Nbs are shown ( n = 1). ( C ) Representative flow cytometry results, showing staining of unmodified HEK293T cells (grey line) or HEK293T cells lentivirally modified to express mouse PD-L1 (293T moPD-L1, red line) with mAbs specific for mouse PD-L1 or Nbs C3, C7, E4 and E2 ( n = 3).

    Techniques Used: Selection, Sequencing, Purification, SPR Assay, Recombinant, Flow Cytometry, Staining, Modification

    5) 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

    Related Articles

    Recombinant:

    Article Title: Peptide Blocking of PD-1/PD-L1 Interaction for Cancer Immunotherapy.
    Article Snippet: In vitro and in vivo experiments showed that one of PD-L1–binding peptides could interfere with PD-1/PD-L1 biological function and, therefore, could be applied as a potential drug for tumor immunotherapy. .. Reagents and cell cultureRecombinant human PD-L1 (cat. #10084-H08H), PD-L2 (cat. #10292-H08H), GM-CSF (cat. #10015-HNAY), IL4 (cat. #11846-HNAE), IL2 (cat. #11848-HNAY1), bFGF (cat. #10014- HNAE), and recombinant mouse PD-L1 (cat. #50010-M08H) were purchased from Sino Biological Inc. Anti-human PD-L1 (cat. #557924, clone MIH1), anti-human PD-1 (cat. #557946, clone MIH4), and anti-human CD4 (cat. # 561841, clone RPAT4) were purchased from BD Biosciences. .. Dynabeads MyOne Streptavidin C1 magnetic beads (cat. #65002), Biotin-XX Microscale Protein Labeling and Detection Kit (cat. #B30756), streptavidin PE (SAPE) conjugates (cat. #S-866), DynaMagSpin Magnet (cat. #12320D), Dynabeads human T-activator CD3/CD28 (cat. #11131D), and Ham's F-12K medium (cat. #21127030) were obtained from Thermo Fisher Scientific.

    Article Title: Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers
    Article Snippet: All Biacore consumables were from GE Healtcare. .. Recombinant mouse PD-L1 protein (Sino Biological #50010-M08H) was used for biopanning and initial enzyme-linked immunosorbant assay (ELISA) screening. .. Mouse or human PD-L1 Fc-fusion proteins (R & Dsystems, 1019-B7 and 156- B7 respectively) were used for calibration-free concentration analysis (CFCA) and affinity estimations of the Nbs in PEs.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers
    Article Snippet: All Biacore consumables were from GE Healtcare. .. Recombinant mouse PD-L1 protein (Sino Biological #50010-M08H) was used for biopanning and initial enzyme-linked immunosorbant assay (ELISA) screening. .. Mouse or human PD-L1 Fc-fusion proteins (R & Dsystems, 1019-B7 and 156- B7 respectively) were used for calibration-free concentration analysis (CFCA) and affinity estimations of the Nbs in PEs.

    Article Title: Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers
    Article Snippet: Mouse or human PD-L1 Fc-fusion proteins (R & Dsystems, 1019-B7 and 156- B7 respectively) were used for calibration-free concentration analysis (CFCA) and affinity estimations of the Nbs in PEs. .. Mouse or human PD-L1 His-tagged proteins (Sino Biological #50010-M08H and #10084-H08H) were used for ELISA screenings of Nbs in PEs and Surface Plasmon Resonance (SPR) affinity determinations on purified Nbs. .. A phycoerythrin labeled antibody specific for mouse PD-L1 (Biolegend, 10F.9G2) or an allophycocyanin labeled antibody specific for human PD-L1 (eBioscience, MIH5) was used in flow cytometry to evaluate PD-L1 expression on cells in vitro.

    SPR Assay:

    Article Title: Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers
    Article Snippet: Mouse or human PD-L1 Fc-fusion proteins (R & Dsystems, 1019-B7 and 156- B7 respectively) were used for calibration-free concentration analysis (CFCA) and affinity estimations of the Nbs in PEs. .. Mouse or human PD-L1 His-tagged proteins (Sino Biological #50010-M08H and #10084-H08H) were used for ELISA screenings of Nbs in PEs and Surface Plasmon Resonance (SPR) affinity determinations on purified Nbs. .. A phycoerythrin labeled antibody specific for mouse PD-L1 (Biolegend, 10F.9G2) or an allophycocyanin labeled antibody specific for human PD-L1 (eBioscience, MIH5) was used in flow cytometry to evaluate PD-L1 expression on cells in vitro.

    Purification:

    Article Title: Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers
    Article Snippet: Mouse or human PD-L1 Fc-fusion proteins (R & Dsystems, 1019-B7 and 156- B7 respectively) were used for calibration-free concentration analysis (CFCA) and affinity estimations of the Nbs in PEs. .. Mouse or human PD-L1 His-tagged proteins (Sino Biological #50010-M08H and #10084-H08H) were used for ELISA screenings of Nbs in PEs and Surface Plasmon Resonance (SPR) affinity determinations on purified Nbs. .. A phycoerythrin labeled antibody specific for mouse PD-L1 (Biolegend, 10F.9G2) or an allophycocyanin labeled antibody specific for human PD-L1 (eBioscience, MIH5) was used in flow cytometry to evaluate PD-L1 expression on cells in vitro.

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    Sino Biological mouse pd l1
    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 <t>SNAP-PD-L1-His</t> 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
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    M2 specifically binds B7.1 and B7.2. ( A ) MEFs transduced to stably express mouse or human B7.1 or B7.2 were verified by specific antibodies ( Upper ) and stained by bio-M2 followed by PE Streptavidin ( Lower ). mAb, monoclonal antibody. ( B , Upper ) Mouse <t>PD-L1/2–transduced</t> CHO cells were stained with bio-M2. CHO-mB7.2 cells served as a positive control. ( B , Lower ).
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    Image Search Results


    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: For , SLB was functionalized by a mixture of 5 nM pMHC-I-His, 2 nM mouse ICAM–His (Sino Biological, 50440-M08H) and either 3 nM mouse PD-L1–His (Sino Biological, 50010-M08H), 3 nM mouse PD-L1–His plus 9 nM mouse CD80–His (Sino Biological, 50446-M08H), or 3 nM mouse PD-L1–His plus 9 nM mouse CD86–His (Sino Biological, 50068-M08H).

    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: For , SLB was functionalized by a mixture of 5 nM pMHC-I-His, 2 nM mouse ICAM–His (Sino Biological, 50440-M08H) and either 3 nM mouse PD-L1–His (Sino Biological, 50010-M08H), 3 nM mouse PD-L1–His plus 9 nM mouse CD80–His (Sino Biological, 50446-M08H), or 3 nM mouse PD-L1–His plus 9 nM mouse CD86–His (Sino Biological, 50068-M08H).

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

    Selection of anti-mouse-PD-L1 specific Nbs ( A ) Amino acid sequence alignment of the purified Nbs C3, C7, E2 and E4. The Nb sequence includes three complementarity-determining regions (CDR 1, 2, 3; indicated in red) and four framework regions (FR1-4, indicated in black). FRs are relatively conserved but CDRs vary widely among Nbs. ( B ) Affinity/kinetics SPR study of purified Nbs interacting with immobilized His-tagged recombinant mouse PD-L1 protein. Sensorgrams of different concentrations of the Nbs are shown ( n  = 1). ( C ) Representative flow cytometry results, showing staining of unmodified HEK293T cells (grey line) or HEK293T cells lentivirally modified to express mouse PD-L1 (293T moPD-L1, red line) with mAbs specific for mouse PD-L1 or Nbs C3, C7, E4 and E2 ( n  = 3).

    Journal: Oncotarget

    Article Title: Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers

    doi: 10.18632/oncotarget.16708

    Figure Lengend Snippet: Selection of anti-mouse-PD-L1 specific Nbs ( A ) Amino acid sequence alignment of the purified Nbs C3, C7, E2 and E4. The Nb sequence includes three complementarity-determining regions (CDR 1, 2, 3; indicated in red) and four framework regions (FR1-4, indicated in black). FRs are relatively conserved but CDRs vary widely among Nbs. ( B ) Affinity/kinetics SPR study of purified Nbs interacting with immobilized His-tagged recombinant mouse PD-L1 protein. Sensorgrams of different concentrations of the Nbs are shown ( n = 1). ( C ) Representative flow cytometry results, showing staining of unmodified HEK293T cells (grey line) or HEK293T cells lentivirally modified to express mouse PD-L1 (293T moPD-L1, red line) with mAbs specific for mouse PD-L1 or Nbs C3, C7, E4 and E2 ( n = 3).

    Article Snippet: Mouse or human PD-L1 His-tagged proteins (Sino Biological #50010-M08H and #10084-H08H) were used for ELISA screenings of Nbs in PEs and Surface Plasmon Resonance (SPR) affinity determinations on purified Nbs.

    Techniques: Selection, Sequencing, Purification, SPR Assay, Recombinant, Flow Cytometry, Staining, Modification

    M2 specifically binds B7.1 and B7.2. ( A ) MEFs transduced to stably express mouse or human B7.1 or B7.2 were verified by specific antibodies ( Upper ) and stained by bio-M2 followed by PE Streptavidin ( Lower ). mAb, monoclonal antibody. ( B , Upper ) Mouse PD-L1/2–transduced CHO cells were stained with bio-M2. CHO-mB7.2 cells served as a positive control. ( B , Lower ).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Cowpox virus encodes a protein that binds B7.1 and B7.2 and subverts T cell costimulation

    doi: 10.1073/pnas.1909414116

    Figure Lengend Snippet: M2 specifically binds B7.1 and B7.2. ( A ) MEFs transduced to stably express mouse or human B7.1 or B7.2 were verified by specific antibodies ( Upper ) and stained by bio-M2 followed by PE Streptavidin ( Lower ). mAb, monoclonal antibody. ( B , Upper ) Mouse PD-L1/2–transduced CHO cells were stained with bio-M2. CHO-mB7.2 cells served as a positive control. ( B , Lower ).

    Article Snippet: The recombinant mouse and human CD28, CTLA4, and PD-L1, all fused with Fc of human IgG1 at the C terminus (except human PD-L1, fused with Fc of mouse IgG1) were from Sino Biological, Inc.

    Techniques: Stable Transfection, Staining, Positive Control