Structured Review

Santa Cruz Biotechnology anti cd81
EGFR is required for HCVcc entry soon after <t>CD81</t> binding but prior to clathrin-mediated endocytosis. (A to E) A time-of-addition experiment (depicted graphically in panel A) was performed in which Huh-7.5 cells were incubated with Jc1-Rluc HCVcc (MOI
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1) Product Images from "Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry"

Article Title: Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry

Journal: Journal of Virology

doi: 10.1128/JVI.00750-12

EGFR is required for HCVcc entry soon after CD81 binding but prior to clathrin-mediated endocytosis. (A to E) A time-of-addition experiment (depicted graphically in panel A) was performed in which Huh-7.5 cells were incubated with Jc1-Rluc HCVcc (MOI
Figure Legend Snippet: EGFR is required for HCVcc entry soon after CD81 binding but prior to clathrin-mediated endocytosis. (A to E) A time-of-addition experiment (depicted graphically in panel A) was performed in which Huh-7.5 cells were incubated with Jc1-Rluc HCVcc (MOI

Techniques Used: Binding Assay, Incubation

TGF-α-mediated internalization of EGFR and CD81. (A) Huh-7.5 cells were treated with 5 nM TGF-α in the presence or absence of 5 μM erlotinib for 15 min at 37°C and fixed with paraformaldehyde, and immunofluorescence staining
Figure Legend Snippet: TGF-α-mediated internalization of EGFR and CD81. (A) Huh-7.5 cells were treated with 5 nM TGF-α in the presence or absence of 5 μM erlotinib for 15 min at 37°C and fixed with paraformaldehyde, and immunofluorescence staining

Techniques Used: Immunofluorescence, Staining

Proposed model for the role of EGFR in HCV entry. HCV binding to CD81 results in cross-linking of CD81 and EGFR kinase activation, which in turn induces cointernalization of HCV-CD81-EGFR. In addition, ligand binding to EGFR activates the receptor and
Figure Legend Snippet: Proposed model for the role of EGFR in HCV entry. HCV binding to CD81 results in cross-linking of CD81 and EGFR kinase activation, which in turn induces cointernalization of HCV-CD81-EGFR. In addition, ligand binding to EGFR activates the receptor and

Techniques Used: Binding Assay, Activation Assay, Ligand Binding Assay

Induction of EGFR activation by HCVcc binding to CD81. For all experiments, total EGFR was immunoprecipitated, and Western blot analyses were performed with an antibody specific for activated EGFR (phosphorylated at Tyr-1068). Total EGFR was detected
Figure Legend Snippet: Induction of EGFR activation by HCVcc binding to CD81. For all experiments, total EGFR was immunoprecipitated, and Western blot analyses were performed with an antibody specific for activated EGFR (phosphorylated at Tyr-1068). Total EGFR was detected

Techniques Used: Activation Assay, Binding Assay, Immunoprecipitation, Western Blot

CD81 cross-linking induces CD81-EGFR colocalization and internalization. (A) Huh-7.5 cells were incubated with FITC-labeled control or anti-CD81 antibodies for 1 h at 4°C or shifted to 37°C for another hour. Cells were fixed and stained
Figure Legend Snippet: CD81 cross-linking induces CD81-EGFR colocalization and internalization. (A) Huh-7.5 cells were incubated with FITC-labeled control or anti-CD81 antibodies for 1 h at 4°C or shifted to 37°C for another hour. Cells were fixed and stained

Techniques Used: Incubation, Labeling, Staining

Huh-7.5 cells were pretreated with erlotinib (○), lapatinib (□), anti-CD81 antibody JS-81 (◆), or anti-E2 antibody AP33 (■) and infected with either Con1/C3 HCVcc (A), Jc1 HCVcc (B), H77 HCVpp (C), Con1 HCVpp (D), or J6CF
Figure Legend Snippet: Huh-7.5 cells were pretreated with erlotinib (○), lapatinib (□), anti-CD81 antibody JS-81 (◆), or anti-E2 antibody AP33 (■) and infected with either Con1/C3 HCVcc (A), Jc1 HCVcc (B), H77 HCVpp (C), Con1 HCVpp (D), or J6CF

Techniques Used: Infection

CD81 cross-linking induces EGFR activation. (A) Huh-7.5 cells were treated with increasing concentrations of anti-CD81 or anti-CLDN1 antibodies (3-fold dilutions starting at 30 μg/ml) for 1 h at 37°C. (B) EGFR activation in Huh-7.5 cells
Figure Legend Snippet: CD81 cross-linking induces EGFR activation. (A) Huh-7.5 cells were treated with increasing concentrations of anti-CD81 or anti-CLDN1 antibodies (3-fold dilutions starting at 30 μg/ml) for 1 h at 37°C. (B) EGFR activation in Huh-7.5 cells

Techniques Used: Activation Assay

2) Product Images from "Cholesterol sensing by CD81 is important for hepatitis C virus entry"

Article Title: Cholesterol sensing by CD81 is important for hepatitis C virus entry

Journal: bioRxiv

doi: 10.1101/542837

Conformational switch mutants modulate HCV entry. We mutated residues D196 and K201 to prevent stabilizing interactions across the EC2-TMD4 hinge. A. We performed five independent MD simulations of WT and D196A K201A CD81 in the presence of cholesterol. Images provide overlaid snapshots from representative simulations. Helix E, TMD4 and cholesterol are color coded by time. For clarity the remaining structure is shown in grey for the t=0ns snapshot only. Structures were orientated using TMD4 as a reference B. The change in angle between Helix E and TMD4, by comparison to the CD81 crystal structure, was measured over time for each D196A K201A simulation; compare to Fig 2B . C. The cumulative time spent in the open conformation for either WT or D196A K201A CD81. D. Huh-7 CD81 KO cells were transduced with lentivectors encoding WT CD81, N18A E219A (cholesterol binding mutant), D196A K201A (open mutant) or K116A D117A (closed mutant). HCV entry was assessed by challenge with a panel of HCVpp (including genotypes 1, 2, 4 and 5). HCVpp infection is shown relative to cells expressing WT CD81. Data from three representative clones and a summary plot of all HCVpp are shown. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). There was no significant difference between N18A E219A and D196A K201A. Error bars indicate standard error of the mean.
Figure Legend Snippet: Conformational switch mutants modulate HCV entry. We mutated residues D196 and K201 to prevent stabilizing interactions across the EC2-TMD4 hinge. A. We performed five independent MD simulations of WT and D196A K201A CD81 in the presence of cholesterol. Images provide overlaid snapshots from representative simulations. Helix E, TMD4 and cholesterol are color coded by time. For clarity the remaining structure is shown in grey for the t=0ns snapshot only. Structures were orientated using TMD4 as a reference B. The change in angle between Helix E and TMD4, by comparison to the CD81 crystal structure, was measured over time for each D196A K201A simulation; compare to Fig 2B . C. The cumulative time spent in the open conformation for either WT or D196A K201A CD81. D. Huh-7 CD81 KO cells were transduced with lentivectors encoding WT CD81, N18A E219A (cholesterol binding mutant), D196A K201A (open mutant) or K116A D117A (closed mutant). HCV entry was assessed by challenge with a panel of HCVpp (including genotypes 1, 2, 4 and 5). HCVpp infection is shown relative to cells expressing WT CD81. Data from three representative clones and a summary plot of all HCVpp are shown. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). There was no significant difference between N18A E219A and D196A K201A. Error bars indicate standard error of the mean.

Techniques Used: Transduction, Binding Assay, Mutagenesis, Infection, Expressing, Clone Assay

Cell surface functionality of CD81 mutants. Huh-7 CD81 KO cells were co-transduced with lentivectors encoding human CD19 and CD81 or empty vector. A. Representative flow cytometry histograms, all samples received CD19 lentivector plus the indicated CD81/control vector. The plot on the left demonstrates CD81 surface expression (i), the right-hand plot displays CD81-dependent trafficking of CD19 to the cell surface (ii). B. CD81 expression on CHO cells confers binding on soluble HCV E2. The plot on the left demonstrates CD81 surface expression (i), the right-hand plot displays sE2 binding to transduced CHO cells (ii). C. Quantification of sE2 binding expressed relative to WT CD81. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). Error bars indicate standard error of the mean.
Figure Legend Snippet: Cell surface functionality of CD81 mutants. Huh-7 CD81 KO cells were co-transduced with lentivectors encoding human CD19 and CD81 or empty vector. A. Representative flow cytometry histograms, all samples received CD19 lentivector plus the indicated CD81/control vector. The plot on the left demonstrates CD81 surface expression (i), the right-hand plot displays CD81-dependent trafficking of CD19 to the cell surface (ii). B. CD81 expression on CHO cells confers binding on soluble HCV E2. The plot on the left demonstrates CD81 surface expression (i), the right-hand plot displays sE2 binding to transduced CHO cells (ii). C. Quantification of sE2 binding expressed relative to WT CD81. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). Error bars indicate standard error of the mean.

Techniques Used: Transduction, Plasmid Preparation, Flow Cytometry, Expressing, Binding Assay

Cholesterol sensing is important for authentic HCV infection. Huh-7 CD81 KO cells were transduced with lentivectors expressing the stated CD81 mutants and were then challenged with J6/JFH HCVcc. A. Representative micrographs of HCVcc infection in transduced cells; DAPI nuclei shown in blue, viral antigen NS5A displayed in orange, scale bar = 100 μ m. B. Quantification of infection, compiled from four independent experiments, data is expressed relative to infection in cells expressing WT CD81. C. Huh-7 Lunet N cells stably expressing the stated CD81 mutants were challenged with a panel of diverse HCVcc bearing the glycoproteins of genotypes 1, 2, 3, 4 and 5. Infection was quantified via a virally encoded luciferase reporter and is expressed relative to WT CD81. Data from three representative clones and a summary plot of all HCVcc are shown. Asterisks indicate statistical significance from WT (n=3, one-way ANOVA, Prism). Error bars indicate standard error of the mean.
Figure Legend Snippet: Cholesterol sensing is important for authentic HCV infection. Huh-7 CD81 KO cells were transduced with lentivectors expressing the stated CD81 mutants and were then challenged with J6/JFH HCVcc. A. Representative micrographs of HCVcc infection in transduced cells; DAPI nuclei shown in blue, viral antigen NS5A displayed in orange, scale bar = 100 μ m. B. Quantification of infection, compiled from four independent experiments, data is expressed relative to infection in cells expressing WT CD81. C. Huh-7 Lunet N cells stably expressing the stated CD81 mutants were challenged with a panel of diverse HCVcc bearing the glycoproteins of genotypes 1, 2, 3, 4 and 5. Infection was quantified via a virally encoded luciferase reporter and is expressed relative to WT CD81. Data from three representative clones and a summary plot of all HCVcc are shown. Asterisks indicate statistical significance from WT (n=3, one-way ANOVA, Prism). Error bars indicate standard error of the mean.

Techniques Used: Infection, Transduction, Expressing, Stable Transfection, Luciferase, Clone Assay

Conformational switch mutants exhibit altered protein interaction networks. A. Volcano plot visualizing differences from co-IPs of Huh-7 Lunet N CD81 WT versus Lunet N control cells (n=4 biological replicates for each cell line). LFQ intensity differences (log2) are plotted against the t-test p value (−logP). Significant interactors were defined by a permutation-based FDR using S0=1 as described [ 94 ]. Reference proteins (CD81, SCARB1, CLDN1, EGFR, TFRC, CAPN5 ITGB and CD151) are highlighted, color coded as in B. B. Mean LFQ intensity differences (log2) of interactors in CD81 co-IP (Huh-7 Lunet N CD81 WT and mutants versus Lunet N control cells). Error bars indicate standard error of the mean (n=4) C. Venn diagrams showing the overlap of significantly enriched proteins found in CD81 co-IPs from WT in grey, N18A E219A (Chl) in orange, D196A K201A (O) in purple and K116A D117A (C) in green. Values below each title indicate significant interactors for each CD81 variant, values in the center of each Venn diagram indicate overlapping interactors.
Figure Legend Snippet: Conformational switch mutants exhibit altered protein interaction networks. A. Volcano plot visualizing differences from co-IPs of Huh-7 Lunet N CD81 WT versus Lunet N control cells (n=4 biological replicates for each cell line). LFQ intensity differences (log2) are plotted against the t-test p value (−logP). Significant interactors were defined by a permutation-based FDR using S0=1 as described [ 94 ]. Reference proteins (CD81, SCARB1, CLDN1, EGFR, TFRC, CAPN5 ITGB and CD151) are highlighted, color coded as in B. B. Mean LFQ intensity differences (log2) of interactors in CD81 co-IP (Huh-7 Lunet N CD81 WT and mutants versus Lunet N control cells). Error bars indicate standard error of the mean (n=4) C. Venn diagrams showing the overlap of significantly enriched proteins found in CD81 co-IPs from WT in grey, N18A E219A (Chl) in orange, D196A K201A (O) in purple and K116A D117A (C) in green. Values below each title indicate significant interactors for each CD81 variant, values in the center of each Venn diagram indicate overlapping interactors.

Techniques Used: Co-Immunoprecipitation Assay, Variant Assay

Conformational switching of CD81 in the absence of cholesterol. We performed five independent 500ns MD simulations of WT CD81 with and without cholesterol. A. Snapshots summarising representative simulations from either condition. The Δ° measurement reflects the change in the angle between helix E of the EC2 and TMD4 (as annotated), by comparison to the CD81 crystal structure. For each snapshot the region from which the measurement was taken is color-coded by time. Cholesterol is shown in red. Structures were orientated using TMD4 as a reference. Examples of the orientation of D196 and K201 are shown as insets. B. The angle between helix E and TMD4 was measured over time for each simulation, 25° was chosen as a threshold to indicate conformational switching. C. The cumulative time spent in the open conformation was calculated across all simulations for either experimental condition. D. The distance between D196 K201 was measured over time for each simulation, the dashed line indicates the distance under which electrostatic interactions and hydrogen bonding occurs (10Å). E. The average distance between D196 and K201 with and without cholesterol, data points represent the mean value for each simulation, asterisk indicates statistical significance (n=5 simulations, unpaired T-test, Prism).
Figure Legend Snippet: Conformational switching of CD81 in the absence of cholesterol. We performed five independent 500ns MD simulations of WT CD81 with and without cholesterol. A. Snapshots summarising representative simulations from either condition. The Δ° measurement reflects the change in the angle between helix E of the EC2 and TMD4 (as annotated), by comparison to the CD81 crystal structure. For each snapshot the region from which the measurement was taken is color-coded by time. Cholesterol is shown in red. Structures were orientated using TMD4 as a reference. Examples of the orientation of D196 and K201 are shown as insets. B. The angle between helix E and TMD4 was measured over time for each simulation, 25° was chosen as a threshold to indicate conformational switching. C. The cumulative time spent in the open conformation was calculated across all simulations for either experimental condition. D. The distance between D196 K201 was measured over time for each simulation, the dashed line indicates the distance under which electrostatic interactions and hydrogen bonding occurs (10Å). E. The average distance between D196 and K201 with and without cholesterol, data points represent the mean value for each simulation, asterisk indicates statistical significance (n=5 simulations, unpaired T-test, Prism).

Techniques Used:

Mutations in the cholesterol binding pocket of CD81 modulate HCV entry. A. Cholesterol (red) is coordinated in the intramembrane cavity of CD81 by hydrogen bonds with inward facing residues N18 and E219. We made various mutations at these sites to disrupt this interaction. B. The cholesterol molecule sits in the centre of an intramembrane binding pocket. In the V68W M72W A108W V212W mutant this space is occupied by tryptophan residues (blue residues). C. The cell surface expression levels of each mutant CD81 was assessed by flow cytometry. D. Huh-7 CD81 KO cells were transduced with lentivector encoding WT CD81 or empty vector control. The cells were surface labelled with anti-CD81 mAb and lysed in Brij-98 detergent buffer. CD81-mAb complexes were pulled-down with protein G beads and associated free cholesterol was measured. Our positive control demonstrates the accuracy of the assay. The dashed line indicates the limit of detection E. We assessed cholesterol association with WT and mutant CD81. Data is expressed relative to WT CD81, asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). A representative western blot demonstrating equivalent levels of CD81 in the whole cell lysate (WCL) and pull-down (IP) F. Huh-7 CD81 KO cells were transduced with lentivectors encoding WT and mutant CD81. HCV entry was assessed by challenge with a panel of HCVpp (including genotypes 1, 2 and 5). HCVpp infection is shown relative to cells expressing WT CD81. Data from three representative clones and a summary plot of all HCVpp are shown. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). Error bars indicate standard error of the mean.
Figure Legend Snippet: Mutations in the cholesterol binding pocket of CD81 modulate HCV entry. A. Cholesterol (red) is coordinated in the intramembrane cavity of CD81 by hydrogen bonds with inward facing residues N18 and E219. We made various mutations at these sites to disrupt this interaction. B. The cholesterol molecule sits in the centre of an intramembrane binding pocket. In the V68W M72W A108W V212W mutant this space is occupied by tryptophan residues (blue residues). C. The cell surface expression levels of each mutant CD81 was assessed by flow cytometry. D. Huh-7 CD81 KO cells were transduced with lentivector encoding WT CD81 or empty vector control. The cells were surface labelled with anti-CD81 mAb and lysed in Brij-98 detergent buffer. CD81-mAb complexes were pulled-down with protein G beads and associated free cholesterol was measured. Our positive control demonstrates the accuracy of the assay. The dashed line indicates the limit of detection E. We assessed cholesterol association with WT and mutant CD81. Data is expressed relative to WT CD81, asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). A representative western blot demonstrating equivalent levels of CD81 in the whole cell lysate (WCL) and pull-down (IP) F. Huh-7 CD81 KO cells were transduced with lentivectors encoding WT and mutant CD81. HCV entry was assessed by challenge with a panel of HCVpp (including genotypes 1, 2 and 5). HCVpp infection is shown relative to cells expressing WT CD81. Data from three representative clones and a summary plot of all HCVpp are shown. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). Error bars indicate standard error of the mean.

Techniques Used: Binding Assay, Mutagenesis, Expressing, Flow Cytometry, Transduction, Plasmid Preparation, Positive Control, Western Blot, Infection, Clone Assay

3) Product Images from "CRC-derived exosomes containing the RNA binding protein HuR promote lung cell proliferation by stabilizing c-Myc mRNA"

Article Title: CRC-derived exosomes containing the RNA binding protein HuR promote lung cell proliferation by stabilizing c-Myc mRNA

Journal: Cancer Biology & Therapy

doi: 10.1080/15384047.2022.2034455

Identification and characterization of HCT116 exosomes. (a) Western blot analysis of HCT116 WT cells and HCT116 HuR KO cells showed the absence of the HuR protein in HCT116 HuR KO cells. (b) Western blot analysis of exosomes isolated from HCT116 WT and HuR KO cell supernatants showing the presence of the traditional exosomal markers CD81, HSP90 and flotillin-1, but absence of calnexin, in both cells lines, with absence of the HuR protein specifically in HuR KO cells. (c and d) Electron micrographs of the exosomes revealed rounded structures with a size of approximately 50–150 nm. The scale bar indicates 100 nm.
Figure Legend Snippet: Identification and characterization of HCT116 exosomes. (a) Western blot analysis of HCT116 WT cells and HCT116 HuR KO cells showed the absence of the HuR protein in HCT116 HuR KO cells. (b) Western blot analysis of exosomes isolated from HCT116 WT and HuR KO cell supernatants showing the presence of the traditional exosomal markers CD81, HSP90 and flotillin-1, but absence of calnexin, in both cells lines, with absence of the HuR protein specifically in HuR KO cells. (c and d) Electron micrographs of the exosomes revealed rounded structures with a size of approximately 50–150 nm. The scale bar indicates 100 nm.

Techniques Used: Western Blot, Isolation

4) Product Images from "Detailed Analysis of Protein Topology of Extracellular Vesicles–Evidence of Unconventional Membrane Protein Orientation"

Article Title: Detailed Analysis of Protein Topology of Extracellular Vesicles–Evidence of Unconventional Membrane Protein Orientation

Journal: Scientific Reports

doi: 10.1038/srep36338

Characterization of EVs and PK-treated EVs. ( a ) Western blot analysis with EV markers, CD81 and TSG101, in 12 fractions from OptiPrep density gradient. ( b ) The particle number in each OptiPrep fraction was analyzed by nanoparticle tracking analysis. ( c ) Cryo-EM images of non-treated and PK-treated EVs. ( d ) Western blot analysis of non-treated and PK-treated EVs with CD81 and beta-actin.
Figure Legend Snippet: Characterization of EVs and PK-treated EVs. ( a ) Western blot analysis with EV markers, CD81 and TSG101, in 12 fractions from OptiPrep density gradient. ( b ) The particle number in each OptiPrep fraction was analyzed by nanoparticle tracking analysis. ( c ) Cryo-EM images of non-treated and PK-treated EVs. ( d ) Western blot analysis of non-treated and PK-treated EVs with CD81 and beta-actin.

Techniques Used: Western Blot

Validation of surface-accessible proteome and inside-out proteins. ( a ) Western blot analysis of surface-accessible and EV proteome. STUB1, GAPDH, Histone H1, and PCNA are surface-accessible proteome, whereas Flotilin 1 and TSG101 are EV proteome. Relative band intensity was measured. ( b ) Flow cytometry of inside-out membrane proteins. EVs were captured by SCAMP3 (left panel) or STX4 (right panel) antibody conjugated beads and then detected with CD63 or CD81. ( c ) The percentage of SCAMP3, STX4, and beta-actin positive EVs were calculated after incubation with or without 0.1% Tween-20.
Figure Legend Snippet: Validation of surface-accessible proteome and inside-out proteins. ( a ) Western blot analysis of surface-accessible and EV proteome. STUB1, GAPDH, Histone H1, and PCNA are surface-accessible proteome, whereas Flotilin 1 and TSG101 are EV proteome. Relative band intensity was measured. ( b ) Flow cytometry of inside-out membrane proteins. EVs were captured by SCAMP3 (left panel) or STX4 (right panel) antibody conjugated beads and then detected with CD63 or CD81. ( c ) The percentage of SCAMP3, STX4, and beta-actin positive EVs were calculated after incubation with or without 0.1% Tween-20.

Techniques Used: Western Blot, Flow Cytometry, Cytometry, Incubation

5) Product Images from "StAR-related lipid transfer domain 11 (STARD11)–mediated ceramide transport mediates extracellular vesicle biogenesis"

Article Title: StAR-related lipid transfer domain 11 (STARD11)–mediated ceramide transport mediates extracellular vesicle biogenesis

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.002587

Palmitate-stimulated EVs are consistent with exosomes. A , EVs isolated from equal number of cells and whole-cell lysates ( WCL ) from immortalized mouse hepatocyte cell line were analyzed by Western blotting. Alix, TSG101, syntenin, and CD81 were used as small EV markers. GAPDH was used as a loading control. B , these expression levels were quantified by densitometry. *, p
Figure Legend Snippet: Palmitate-stimulated EVs are consistent with exosomes. A , EVs isolated from equal number of cells and whole-cell lysates ( WCL ) from immortalized mouse hepatocyte cell line were analyzed by Western blotting. Alix, TSG101, syntenin, and CD81 were used as small EV markers. GAPDH was used as a loading control. B , these expression levels were quantified by densitometry. *, p

Techniques Used: Isolation, Western Blot, Expressing

6) Product Images from "Natural-Killer-Derived Extracellular Vesicles: Immune Sensors and Interactors"

Article Title: Natural-Killer-Derived Extracellular Vesicles: Immune Sensors and Interactors

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.00262

Morphological characterization of natural killer cell-derived extracellular vesicles (NKEVs). (A,B) Nanoparticle tracking analysis (NTA) analysis of NK-cell-derived microvesicle (NKMV) and NKExo. Representative spectra are shown, and mean, mode, and particles number/ml ([]/ml) are reported. (C,D) Scanning electron microscopy analysis of NKMV (C) and NKExo (D) . Bars, 500 nm. (E,F) Transmission electron microscopy analysis of NKMV (E) and NKExo (F) . Bars, 200 nm. (G–J) Immunoelectron microscopy combined with positive/negative contrast method of NKMV (G,H) and NKExo (I,J) revealed the presence of CD81 (G,I) and CD56 (H,J) . Bars, 100 nm.
Figure Legend Snippet: Morphological characterization of natural killer cell-derived extracellular vesicles (NKEVs). (A,B) Nanoparticle tracking analysis (NTA) analysis of NK-cell-derived microvesicle (NKMV) and NKExo. Representative spectra are shown, and mean, mode, and particles number/ml ([]/ml) are reported. (C,D) Scanning electron microscopy analysis of NKMV (C) and NKExo (D) . Bars, 500 nm. (E,F) Transmission electron microscopy analysis of NKMV (E) and NKExo (F) . Bars, 200 nm. (G–J) Immunoelectron microscopy combined with positive/negative contrast method of NKMV (G,H) and NKExo (I,J) revealed the presence of CD81 (G,I) and CD56 (H,J) . Bars, 100 nm.

Techniques Used: Derivative Assay, Electron Microscopy, Transmission Assay, Immuno-Electron Microscopy

7) Product Images from "Exosomal Annexin A2 Promotes Angiogenesis and Breast Cancer Metastasis"

Article Title: Exosomal Annexin A2 Promotes Angiogenesis and Breast Cancer Metastasis

Journal: Molecular cancer research : MCR

doi: 10.1158/1541-7786.MCR-16-0163

Exo-AnxA2 promotes angiogenesis via tPA and activates macrophages, leading to secretion of IL-6 and TNF-alpha (A) Endothelial tube formation assay showing the role of tPA in the pro-angiogenic effect of exo-AnxA2 (n=2). Quantification of the number of meshes/field (B) and number of branch points/field (C). Exo-AnxA2 promotes breast cancer metastasis to lungs . Western blot analysis of WCL (D) and exosomal lysates (E) showing knockdown of AnxA2 (n=2); GAPDH and CD81 were used as loading controls for (D) and (E), respectively. Quantification of exosomal AnxA2 (F). G) Quantification of BLI of PBS-, 231-AnxA2KD-Exo-, or 231-Control-Exo-primed animals 35 days after challenge with MDA-MB-231-luc cells (lateral tail vein injection), showing differences in the extent of lung metastasis (n=8). Fold change in photon flux to PBS-primed animals is shown. H) Quantification of bioluminescence (BLI) of PBS-, 4175-AnxA2KD-Exo-, or 4175-Control-Exo-primed animals 44 days after challenge with MDA-MB-4175-luc cells (intracardiac; ic) showing differences in the extent of lung metastasis (n=8). Fold change in photon flux to PBS-primed animals is shown. I) India ink staining of the excised lungs from MDA-MB-231-luc cell-injected animals, showing the number of metastatic nodules. Quantification of the number of metastatic lung nodules with MDA-MB-231-luc (tail vein injection) treatment (J) and MDA-MB-4175-luc (ic) treatment (K). Fold change compared to PBS-primed animals is shown. (*) p
Figure Legend Snippet: Exo-AnxA2 promotes angiogenesis via tPA and activates macrophages, leading to secretion of IL-6 and TNF-alpha (A) Endothelial tube formation assay showing the role of tPA in the pro-angiogenic effect of exo-AnxA2 (n=2). Quantification of the number of meshes/field (B) and number of branch points/field (C). Exo-AnxA2 promotes breast cancer metastasis to lungs . Western blot analysis of WCL (D) and exosomal lysates (E) showing knockdown of AnxA2 (n=2); GAPDH and CD81 were used as loading controls for (D) and (E), respectively. Quantification of exosomal AnxA2 (F). G) Quantification of BLI of PBS-, 231-AnxA2KD-Exo-, or 231-Control-Exo-primed animals 35 days after challenge with MDA-MB-231-luc cells (lateral tail vein injection), showing differences in the extent of lung metastasis (n=8). Fold change in photon flux to PBS-primed animals is shown. H) Quantification of bioluminescence (BLI) of PBS-, 4175-AnxA2KD-Exo-, or 4175-Control-Exo-primed animals 44 days after challenge with MDA-MB-4175-luc cells (intracardiac; ic) showing differences in the extent of lung metastasis (n=8). Fold change in photon flux to PBS-primed animals is shown. I) India ink staining of the excised lungs from MDA-MB-231-luc cell-injected animals, showing the number of metastatic nodules. Quantification of the number of metastatic lung nodules with MDA-MB-231-luc (tail vein injection) treatment (J) and MDA-MB-4175-luc (ic) treatment (K). Fold change compared to PBS-primed animals is shown. (*) p

Techniques Used: Endothelial Tube Formation Assay, Western Blot, Multiple Displacement Amplification, Injection, Staining

Characterization of exosomes (A–F) A) Western blot analysis of the different protein levels in the exosomes collected from the MCF10A-, MCF10AT-, and MCF10CA1a-conditioned media. CD81 was used as a loading control (n=3). The Coomassie band confirms equal loading. B) Quantification of the Western blots. Fold change to CD81 is shown. C) Atomic force microscopy (AFM) analysis of MCF10A exosomes and MCF10CA1a exosomes. AnxA2, calnexin, or CD63 immunoreactivity was identified by 25-nm gold nanoparticles. D) Quantification of the AFM data representing the number of secondary gold nanoparticles per field. Four independent fields were counted. E) Surface topology analysis of the exosomes using the AFM NT-MDT software (n=2). (*) p
Figure Legend Snippet: Characterization of exosomes (A–F) A) Western blot analysis of the different protein levels in the exosomes collected from the MCF10A-, MCF10AT-, and MCF10CA1a-conditioned media. CD81 was used as a loading control (n=3). The Coomassie band confirms equal loading. B) Quantification of the Western blots. Fold change to CD81 is shown. C) Atomic force microscopy (AFM) analysis of MCF10A exosomes and MCF10CA1a exosomes. AnxA2, calnexin, or CD63 immunoreactivity was identified by 25-nm gold nanoparticles. D) Quantification of the AFM data representing the number of secondary gold nanoparticles per field. Four independent fields were counted. E) Surface topology analysis of the exosomes using the AFM NT-MDT software (n=2). (*) p

Techniques Used: Western Blot, Microscopy, Software

8) Product Images from "The Innate Immune Factor Apolipoprotein L1 Restricts HIV-1 Infection"

Article Title: The Innate Immune Factor Apolipoprotein L1 Restricts HIV-1 Infection

Journal: Journal of Virology

doi: 10.1128/JVI.02828-13

APOL1 depletes intracellular Vif by lysosomal degradation and stimulating its secretion in microvesicles. (A) 293T cells were transfected with Vif-expressing pNL-A1 vector alone or in combination with APOL1 expression vector. Five hours after transfection, the cells were treated with E64d (10 μg/ml) and pepstatin A (10 μg/ml). After 24 h, whole-cell lysates and pelleted microvesicles were separated by 10% SDS-PAGE and analyzed by Western blotting for the expression of Vif, APOL1, and microvesicle markers Alix and CD81. Secretion of microvesicles was inhibited by incubation of transfected cells with the calcium chelator BAPTA-AM (10 μM) for 16 h. (B) Depletion of Rab7A with siRNA inhibited lysosomal degradation of Vif and potentiated APOL1-mediated release of Vif in microvesicles. 293T cells were transfected twice with Rab7A siRNA, followed by transfection with pNL-A1 alone or with APOL1 vector. After 24 h, whole-cell lysates and pelleted microvesicles were separated by 10% SDS-PAGE and analyzed by Western blotting for the expression of Vif, APOL1, and Rab7 and microvesicle markers Alix and Tsg101. Co, control.
Figure Legend Snippet: APOL1 depletes intracellular Vif by lysosomal degradation and stimulating its secretion in microvesicles. (A) 293T cells were transfected with Vif-expressing pNL-A1 vector alone or in combination with APOL1 expression vector. Five hours after transfection, the cells were treated with E64d (10 μg/ml) and pepstatin A (10 μg/ml). After 24 h, whole-cell lysates and pelleted microvesicles were separated by 10% SDS-PAGE and analyzed by Western blotting for the expression of Vif, APOL1, and microvesicle markers Alix and CD81. Secretion of microvesicles was inhibited by incubation of transfected cells with the calcium chelator BAPTA-AM (10 μM) for 16 h. (B) Depletion of Rab7A with siRNA inhibited lysosomal degradation of Vif and potentiated APOL1-mediated release of Vif in microvesicles. 293T cells were transfected twice with Rab7A siRNA, followed by transfection with pNL-A1 alone or with APOL1 vector. After 24 h, whole-cell lysates and pelleted microvesicles were separated by 10% SDS-PAGE and analyzed by Western blotting for the expression of Vif, APOL1, and Rab7 and microvesicle markers Alix and Tsg101. Co, control.

Techniques Used: Transfection, Expressing, Plasmid Preparation, SDS Page, Western Blot, Incubation

9) Product Images from "Engineering of extracellular vesicles for display of protein biotherapeutics"

Article Title: Engineering of extracellular vesicles for display of protein biotherapeutics

Journal: bioRxiv

doi: 10.1101/2020.06.14.149823

Multimeric decoy receptor EV sorting protein chimera is functionalised on several EV subpopulations. A) Engineered EVs displaying IL6ST purified from MSC cells stably expressing the optimised IL6STΔ-LZ-NST display construct, evaluated for IL6/sIL6R decoy in an in vitro cell assay respondent to IL6/sIL6R induced STAT3 activation. EVs purified from MSC stably expressing Ctrl construct were used as control. Data were normalized to control cells treated with IL6/sIL6R (5 ng/ml). B) Engineered EVs displaying TNFR1 purified from MSC cells stably expressing the optimized TNFR1ΔΔ-FDN-NST display construct, evaluated for TNFα decoy in an in vitro cell assay responsive to TNFα induced NF-κB activation. EVs purified from MSC stably expressing Ctrl construct were used as control. Data were normalized to control cells treated with TNFα (5 ng/ml) treated cells. C) WB of MSC TNFR1ΔΔ-FDN-NST, IL6STΔ-LZ-NST and Ctrl cells and EVs indicating the presence of classical EV markers; ALIX (96 kDa), TSG101 (44 kDa) and absence of Calnexin (67 kDa) in the isolated EVs. The WB results further demonstrate the presence of respective His-tagged decoy receptors; TNFR1ΔΔ-FDN-NST (48 kDa) and IL6STΔ-LZ-NST (94 kDa) both on cells and EVs. D) Transmission electron microscopy of MSC TNFR1ΔΔ-FDN-NST, IL6STΔ-LZ-NST and Ctrl-EVs with nanogold labelled antibody staining of respective decoy receptor indicated by white arrows. E) Schematic illustration showing the workflow of the multiplex bead-based flow cytometry assay. Isolated EVs incubated with up to 39 different bead populations coated with different capture antibodies, which are distinguishable by flow cytometry due to their different fluorescence intensities. EVs captured by the different beads are detected with detection antibodies either against PAN (CD63-APC, CD81-APC and CD9-APC), mIL6ST-APC, or hTNFR1-APC. F-H) Characterization of EV surface protein composition by using F) anti-PAN (CD63, CD81 and CD9), G) anti-hTNFR1 and H) anti-mIL6ST detection antibodies in multiplex bead-based assays to confirm marker co-expression on MSC TNFR1ΔΔ-FDN-NST, IL6STΔ-LZ-NST and ctrl EVs. Data represented as background corrected median APC fluorescence intensity determined by flow cytometry of EVs bound to respective capture beads and upon using APC labelled detection antibody. A, B , Error bars, s.d. ( n = 3), **** P
Figure Legend Snippet: Multimeric decoy receptor EV sorting protein chimera is functionalised on several EV subpopulations. A) Engineered EVs displaying IL6ST purified from MSC cells stably expressing the optimised IL6STΔ-LZ-NST display construct, evaluated for IL6/sIL6R decoy in an in vitro cell assay respondent to IL6/sIL6R induced STAT3 activation. EVs purified from MSC stably expressing Ctrl construct were used as control. Data were normalized to control cells treated with IL6/sIL6R (5 ng/ml). B) Engineered EVs displaying TNFR1 purified from MSC cells stably expressing the optimized TNFR1ΔΔ-FDN-NST display construct, evaluated for TNFα decoy in an in vitro cell assay responsive to TNFα induced NF-κB activation. EVs purified from MSC stably expressing Ctrl construct were used as control. Data were normalized to control cells treated with TNFα (5 ng/ml) treated cells. C) WB of MSC TNFR1ΔΔ-FDN-NST, IL6STΔ-LZ-NST and Ctrl cells and EVs indicating the presence of classical EV markers; ALIX (96 kDa), TSG101 (44 kDa) and absence of Calnexin (67 kDa) in the isolated EVs. The WB results further demonstrate the presence of respective His-tagged decoy receptors; TNFR1ΔΔ-FDN-NST (48 kDa) and IL6STΔ-LZ-NST (94 kDa) both on cells and EVs. D) Transmission electron microscopy of MSC TNFR1ΔΔ-FDN-NST, IL6STΔ-LZ-NST and Ctrl-EVs with nanogold labelled antibody staining of respective decoy receptor indicated by white arrows. E) Schematic illustration showing the workflow of the multiplex bead-based flow cytometry assay. Isolated EVs incubated with up to 39 different bead populations coated with different capture antibodies, which are distinguishable by flow cytometry due to their different fluorescence intensities. EVs captured by the different beads are detected with detection antibodies either against PAN (CD63-APC, CD81-APC and CD9-APC), mIL6ST-APC, or hTNFR1-APC. F-H) Characterization of EV surface protein composition by using F) anti-PAN (CD63, CD81 and CD9), G) anti-hTNFR1 and H) anti-mIL6ST detection antibodies in multiplex bead-based assays to confirm marker co-expression on MSC TNFR1ΔΔ-FDN-NST, IL6STΔ-LZ-NST and ctrl EVs. Data represented as background corrected median APC fluorescence intensity determined by flow cytometry of EVs bound to respective capture beads and upon using APC labelled detection antibody. A, B , Error bars, s.d. ( n = 3), **** P

Techniques Used: Purification, Stable Transfection, Expressing, Construct, In Vitro, Activation Assay, Western Blot, Isolation, Transmission Assay, Electron Microscopy, Staining, Multiplex Assay, Flow Cytometry, Incubation, Fluorescence, Marker

Dual functionalized Engineered EVs protect against intestinal inflammation. A) Imaging flow cytometry analysis (IFCM) with dot plots and example event images in the double positive (DP) gate of MSC TNFR1ΔΔ-FDN-NST and MSC double decoy EVs stained with mIL6ST APC conjugated and hTNFR1 PE conjugated antibody. PBS + antibodies were used for background adjustment and for determining the gating strategy. B) Percentage of detected events positive for either hTNFR1 or mIL6ST or both in Imaging flow cytometry analysis of MSC TNFR1ΔΔ-FDN-NST and MSC double decoy EVs stained with mIL6ST APC conjugated and hTNFR1 PE conjugated antibody. Percentage values determined from objects/ml in different gates. C) Transmission electron microscopy of double decoy EVs with nanogold labelled antibody staining of hTNFR1 (10 nm) and mIL6ST (5 nm). D) Multiplex EV surface characterization of PAN (CD63, CD81, and CD9) positive, hTNFR1 positive and mIL6ST positive population in MSC double decoy EVs and MSC Ctrl EVs. Data represented as background corrected median APC fluorescence intensity determined by flow cytometry of EVs bound different capture beads and upon using APC labelled detection antibody. E) Engineered EVs displaying TNFR1 purified from MSC cells stably expressing either the optimized TNFR1ΔΔ-FDN-NST display construct or TNFR1ΔΔ-FDN-NST and IL6STΔ-LZ-NST construct, evaluated for TNFα decoy in an in vitro cell assay responsive to TNFα induced NF-κB activation. EVs purified from MSC stably expressing either the IL6STΔ-LZ-NST display construct or Ctrl construct were used as control. Data were normalized to control cells treated with TNFα (5 ng/ml). F) Engineered EVs displaying IL6ST purified from MSC cells stably expressing either the optimized IL6STΔ-LZ-NST display construct or TNFR1ΔΔ-FDN-NST, evaluated for IL6/sIL6R decoy in an in vitro cell assay responsive to IL6/sIL6R induced STAT3 activation. EVs purified from MSC stably expressing either the TNFR1ΔΔ-FDN-NST display construct or Ctrl construct were used as control. Data were normalized to control cells treated with IL6/sIL6R (5 ng/ml). G) Schematic of the treatment protocol for double decoy EVs in TNBS induced colitis. H) Percent change in relative bodyweight to initial weight over the disease course and I) survival curve in mice induced with colitis by intrarectal injection of TNBS and treated I.V with either 3×10 11 MSC double decoy EVs ( n =13), 3×10 10 MSC double decoy EVs ( n =15), 10 µ g Tocilizumab and 1 µ g Etanercept ( n =14), or saline (n=15) 24 hours post disease induction. E, F , Error bars, s.d. ( n =3). **** P
Figure Legend Snippet: Dual functionalized Engineered EVs protect against intestinal inflammation. A) Imaging flow cytometry analysis (IFCM) with dot plots and example event images in the double positive (DP) gate of MSC TNFR1ΔΔ-FDN-NST and MSC double decoy EVs stained with mIL6ST APC conjugated and hTNFR1 PE conjugated antibody. PBS + antibodies were used for background adjustment and for determining the gating strategy. B) Percentage of detected events positive for either hTNFR1 or mIL6ST or both in Imaging flow cytometry analysis of MSC TNFR1ΔΔ-FDN-NST and MSC double decoy EVs stained with mIL6ST APC conjugated and hTNFR1 PE conjugated antibody. Percentage values determined from objects/ml in different gates. C) Transmission electron microscopy of double decoy EVs with nanogold labelled antibody staining of hTNFR1 (10 nm) and mIL6ST (5 nm). D) Multiplex EV surface characterization of PAN (CD63, CD81, and CD9) positive, hTNFR1 positive and mIL6ST positive population in MSC double decoy EVs and MSC Ctrl EVs. Data represented as background corrected median APC fluorescence intensity determined by flow cytometry of EVs bound different capture beads and upon using APC labelled detection antibody. E) Engineered EVs displaying TNFR1 purified from MSC cells stably expressing either the optimized TNFR1ΔΔ-FDN-NST display construct or TNFR1ΔΔ-FDN-NST and IL6STΔ-LZ-NST construct, evaluated for TNFα decoy in an in vitro cell assay responsive to TNFα induced NF-κB activation. EVs purified from MSC stably expressing either the IL6STΔ-LZ-NST display construct or Ctrl construct were used as control. Data were normalized to control cells treated with TNFα (5 ng/ml). F) Engineered EVs displaying IL6ST purified from MSC cells stably expressing either the optimized IL6STΔ-LZ-NST display construct or TNFR1ΔΔ-FDN-NST, evaluated for IL6/sIL6R decoy in an in vitro cell assay responsive to IL6/sIL6R induced STAT3 activation. EVs purified from MSC stably expressing either the TNFR1ΔΔ-FDN-NST display construct or Ctrl construct were used as control. Data were normalized to control cells treated with IL6/sIL6R (5 ng/ml). G) Schematic of the treatment protocol for double decoy EVs in TNBS induced colitis. H) Percent change in relative bodyweight to initial weight over the disease course and I) survival curve in mice induced with colitis by intrarectal injection of TNBS and treated I.V with either 3×10 11 MSC double decoy EVs ( n =13), 3×10 10 MSC double decoy EVs ( n =15), 10 µ g Tocilizumab and 1 µ g Etanercept ( n =14), or saline (n=15) 24 hours post disease induction. E, F , Error bars, s.d. ( n =3). **** P

Techniques Used: Imaging, Flow Cytometry, Staining, Transmission Assay, Electron Microscopy, Multiplex Assay, Fluorescence, Purification, Stable Transfection, Expressing, Construct, In Vitro, Activation Assay, Mouse Assay, Injection

10) Product Images from "Exosomes from Hepatitis C Infected Patients Transmit HCV Infection and Contain Replication Competent Viral RNA in Complex with Ago2-miR122-HSP90"

Article Title: Exosomes from Hepatitis C Infected Patients Transmit HCV Infection and Contain Replication Competent Viral RNA in Complex with Ago2-miR122-HSP90

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004424

CD81 deficiency or anti-HCV E2 antibody treatment does not block effective HCV transmission by exosomes. (A) CD81 deficient Huh7.25 and (B) Huh7.0 cells were infected with HCV J6/JFH-1 virus or exosomes from culture supernatants of HCV J6/JFH-1 infected Huh7.5 cells as indicated. Viruses and exosomes were washed off after 8 h of co-culture and fresh medium added to the cells and cultured at 37°C for another 40 h. Total RNA was then extracted from cells and analysed for HCV RNA by RT-qPCR. An MOI of 1 of infectious HCV virus and infectious HCV-exosomes were used for all infections. Input exosome samples were appropriately matched by using 100 ng of total input exosome RNA and 100 ng of total infected cell RNA for HCV RNA comparison after infection to allow valid comparison. (C) Huh7.5 cells were infected with HCV J6/JFH-1 virus or with HCV exosomes from culture supernatants of HCV J6/JFH-1 infected Huh7.5 cells with anti-HCV E2 antibody pre-treatment (1∶50, 1∶100 dilutions) or not as indicated. Viruses and exosomes were washed off after 8 h of co-culture and fresh medium was added to cells followed by 40 h further culture at 37°C. Total RNA and protein was then extracted from cells and analyzed by quantitative RT-PCR for HCV RNA. Results presented are representative of 3 independent experiments expressed as mean + SEM, p
Figure Legend Snippet: CD81 deficiency or anti-HCV E2 antibody treatment does not block effective HCV transmission by exosomes. (A) CD81 deficient Huh7.25 and (B) Huh7.0 cells were infected with HCV J6/JFH-1 virus or exosomes from culture supernatants of HCV J6/JFH-1 infected Huh7.5 cells as indicated. Viruses and exosomes were washed off after 8 h of co-culture and fresh medium added to the cells and cultured at 37°C for another 40 h. Total RNA was then extracted from cells and analysed for HCV RNA by RT-qPCR. An MOI of 1 of infectious HCV virus and infectious HCV-exosomes were used for all infections. Input exosome samples were appropriately matched by using 100 ng of total input exosome RNA and 100 ng of total infected cell RNA for HCV RNA comparison after infection to allow valid comparison. (C) Huh7.5 cells were infected with HCV J6/JFH-1 virus or with HCV exosomes from culture supernatants of HCV J6/JFH-1 infected Huh7.5 cells with anti-HCV E2 antibody pre-treatment (1∶50, 1∶100 dilutions) or not as indicated. Viruses and exosomes were washed off after 8 h of co-culture and fresh medium was added to cells followed by 40 h further culture at 37°C. Total RNA and protein was then extracted from cells and analyzed by quantitative RT-PCR for HCV RNA. Results presented are representative of 3 independent experiments expressed as mean + SEM, p

Techniques Used: Blocking Assay, Transmission Assay, Infection, Co-Culture Assay, Cell Culture, Quantitative RT-PCR

Schematics of exosome mediated HCV transmission and possible therpaeutic strategies. HCV infected hepatocytes form multivesicular bodies (MVB) by taking up bits of the cytoplasm and its contents (proteins [HSP90], miRNA [miR-122] and RNAs [HCV RNA]) into membrane-bound vesicles. These MVB in HCV infected hepatocytes then fuse with the plasma membrane and release their contents, including numerous small vesicles (exosomes), outside the cell. Exosomes released from HCV infected hepatocytes contain replication competent HCV RNA in complex with miR-122, Ago2, and HSP90. Further, exosomes released from HCV infected hepatocytes are capable of CD81-independent HCV transmission to a naïve hepatocyte. The use of HSP90, miR-122, proton pump and Vacuolar-type H+-ATPase inhibitors can significantly limit the capacity of HCV transmission by exosomes.
Figure Legend Snippet: Schematics of exosome mediated HCV transmission and possible therpaeutic strategies. HCV infected hepatocytes form multivesicular bodies (MVB) by taking up bits of the cytoplasm and its contents (proteins [HSP90], miRNA [miR-122] and RNAs [HCV RNA]) into membrane-bound vesicles. These MVB in HCV infected hepatocytes then fuse with the plasma membrane and release their contents, including numerous small vesicles (exosomes), outside the cell. Exosomes released from HCV infected hepatocytes contain replication competent HCV RNA in complex with miR-122, Ago2, and HSP90. Further, exosomes released from HCV infected hepatocytes are capable of CD81-independent HCV transmission to a naïve hepatocyte. The use of HSP90, miR-122, proton pump and Vacuolar-type H+-ATPase inhibitors can significantly limit the capacity of HCV transmission by exosomes.

Techniques Used: Transmission Assay, Infection

Exosomes from HCV J6/JFH-1 infected Huh7.5 cells and HCV patients mediate effective HCV transmission in the presence of anti-CD81, anti-SB-RI or anti-APOE antibody. (A B) Huh7.5 cells were infected with HCV J6/JFH-1 virus or with exosomes from culture supernatants of HCV J6/JFH-1 infected Huh7.5 cells with CD81 antibody pre-treatment (1∶50 dilution) or not as indicated. Viruses and exosomes were washed off after 8 h of co-culture and fresh medium was added to cells followed by 40 h further culture at 37°C. Total RNA and protein was then extracted from cells and analyzed by (A) quantitative RT-PCR for HCV RNA and by (B) western blotting for HCV NS3 protein. (C D) Primary human hepatocytes were infected with either serum exosomes or free HCV viruses from HCV treatment naïve patients; exosomes from Huh7.5-HCV J6/JFH-1 culture supernatants; HCV J6/JFH-1 virus along with anti-CD81 antibody pre-treatment for one hour before infection or not as indicated for 48 h. Total RNA and protein was then extracted from cells and analysed for HCV RNA by quantitative RT-PCR and by western blot for HCV NS3 protein. An MOI of 1 of infectious HCV virus and infectious HCV-exosomes were used for all infections. (E F) Huh7.5 cells were infected with either HCV J6/JFH-1 exosomes or free HCV J6/JFH-1 viruses for 48 h after prior treatment of cells with anti-SB-RI antibody (1∶50 or 1∶100 dilution) ( Fig. 5E ) or anti-APOE antibody (1∶50 dilution) ( Fig. 5F ) at concentrations indicated for one hour before infections. Total RNA and protein was then extracted from cells and analysed for HCV RNA by quantitative RT-PCR and by western blot for HCV NS3 protein. An MOI of 1 of infectious HCV virus and infectious HCV-exosomes were used for all infections. Results presented are representative of 3 independent experiments expressed as mean + SEM, p
Figure Legend Snippet: Exosomes from HCV J6/JFH-1 infected Huh7.5 cells and HCV patients mediate effective HCV transmission in the presence of anti-CD81, anti-SB-RI or anti-APOE antibody. (A B) Huh7.5 cells were infected with HCV J6/JFH-1 virus or with exosomes from culture supernatants of HCV J6/JFH-1 infected Huh7.5 cells with CD81 antibody pre-treatment (1∶50 dilution) or not as indicated. Viruses and exosomes were washed off after 8 h of co-culture and fresh medium was added to cells followed by 40 h further culture at 37°C. Total RNA and protein was then extracted from cells and analyzed by (A) quantitative RT-PCR for HCV RNA and by (B) western blotting for HCV NS3 protein. (C D) Primary human hepatocytes were infected with either serum exosomes or free HCV viruses from HCV treatment naïve patients; exosomes from Huh7.5-HCV J6/JFH-1 culture supernatants; HCV J6/JFH-1 virus along with anti-CD81 antibody pre-treatment for one hour before infection or not as indicated for 48 h. Total RNA and protein was then extracted from cells and analysed for HCV RNA by quantitative RT-PCR and by western blot for HCV NS3 protein. An MOI of 1 of infectious HCV virus and infectious HCV-exosomes were used for all infections. (E F) Huh7.5 cells were infected with either HCV J6/JFH-1 exosomes or free HCV J6/JFH-1 viruses for 48 h after prior treatment of cells with anti-SB-RI antibody (1∶50 or 1∶100 dilution) ( Fig. 5E ) or anti-APOE antibody (1∶50 dilution) ( Fig. 5F ) at concentrations indicated for one hour before infections. Total RNA and protein was then extracted from cells and analysed for HCV RNA by quantitative RT-PCR and by western blot for HCV NS3 protein. An MOI of 1 of infectious HCV virus and infectious HCV-exosomes were used for all infections. Results presented are representative of 3 independent experiments expressed as mean + SEM, p

Techniques Used: Infection, Transmission Assay, Co-Culture Assay, Quantitative RT-PCR, Western Blot

11) Product Images from "Influence of species and processing parameters on recovery and content of brain tissue-derived extracellular vesicles"

Article Title: Influence of species and processing parameters on recovery and content of brain tissue-derived extracellular vesicles

Journal: Journal of Extracellular Vesicles

doi: 10.1080/20013078.2020.1785746

Characterization of human bdEVs obtained by three combinations of methods . (a) Particle concentration of human bdEVs separated by three combinations of methods was measured by NTA (Particle Metrix). Particle concentration for each group was normalized by tissue mass (per 100 mg). (b) Protein concentration of human bdEVs separated by different methods and measured by BCA protein assay (per 100 mg tissue). (c) Ratio of particles to protein (particles/µg). (a–c): Data are presented as the mean with range. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 by two-tailed Welch’s t-test. (d) bdEVs were visualized by negative staining transmission electron microscopy (scale bar = 100 nm). TEM is representative of five images taken of each fraction from three independent human tissue samples. (e) Western blot analysis of GM130, calnexin, CD81, CD63, and syntenin associated with BH and EV fractions. WB are representative of three independent human tissue EV separations from the SDGU and SEC+UC methods, and one independent human tissue EV separation from the SEC+UF method.
Figure Legend Snippet: Characterization of human bdEVs obtained by three combinations of methods . (a) Particle concentration of human bdEVs separated by three combinations of methods was measured by NTA (Particle Metrix). Particle concentration for each group was normalized by tissue mass (per 100 mg). (b) Protein concentration of human bdEVs separated by different methods and measured by BCA protein assay (per 100 mg tissue). (c) Ratio of particles to protein (particles/µg). (a–c): Data are presented as the mean with range. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 by two-tailed Welch’s t-test. (d) bdEVs were visualized by negative staining transmission electron microscopy (scale bar = 100 nm). TEM is representative of five images taken of each fraction from three independent human tissue samples. (e) Western blot analysis of GM130, calnexin, CD81, CD63, and syntenin associated with BH and EV fractions. WB are representative of three independent human tissue EV separations from the SDGU and SEC+UC methods, and one independent human tissue EV separation from the SEC+UF method.

Techniques Used: Concentration Assay, Protein Concentration, Bicinchoninic Acid Protein Assay, Two Tailed Test, Negative Staining, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Western Blot

12) Product Images from "ADAM8-Dependent Extracellular Signaling in the Tumor Microenvironment Involves Regulated Release of Lipocalin 2 and MMP-9"

Article Title: ADAM8-Dependent Extracellular Signaling in the Tumor Microenvironment Involves Regulated Release of Lipocalin 2 and MMP-9

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms23041976

ADAM8 is secreted by Panc89 hA8 WT-derived extracellular vesicles (EV). ( A ) The histogram shows the particle size distribution of EVs isolated from Panc89 cells (analyzed by NanoFCM). ( B ) Electron microscopy of Panc89 hA8 WT-derived EVs demonstrates the successful isolation of EVs; scale bar, 100 nm. Representative Western blot of EVs derived either from Panc89 hA8 WT or KO cells, and cell lysate (CL) of Panc89 hA8 WT cells is shown in ( C ). ADAM8 can be detected as active and remnant ADAM8 in EVs. The negative control Calnexin was not detectable in isolated EVs. The measured activity of Panc89 hA8 WT- and KO-derived EVs on PepDAB #13 is displayed in ( D ) and is upregulated in Panc89 hA8 WT-derived EVs ( n = 2). ( E ) Representative images of immunofluorescence staining of Panc89 hA8 rescue cells with Hoechst dye (upper left), TSG101 (green; upper right), and ADAM8 (red; lower left). Merged images are displayed in the lower right and show that TSG101 shows little or no co-localization with ADAM8. Scale bar, 50 μm. ( F ) shows the detection of ADAM8, Flotillin-1, and CD81 via Western blot of Panc89 hA8 WT CL and EV preparations isolated from Panc89 hA8 WT, Panc89 hA8 rescue, Panc89 hA8 ΔCD rescue, and Panc89 hA8 KO cells. ADAM8 is detectable in all EV preparations except in EVs isolated from hA8 KO cells. Data are presented as mean values ± S.D.
Figure Legend Snippet: ADAM8 is secreted by Panc89 hA8 WT-derived extracellular vesicles (EV). ( A ) The histogram shows the particle size distribution of EVs isolated from Panc89 cells (analyzed by NanoFCM). ( B ) Electron microscopy of Panc89 hA8 WT-derived EVs demonstrates the successful isolation of EVs; scale bar, 100 nm. Representative Western blot of EVs derived either from Panc89 hA8 WT or KO cells, and cell lysate (CL) of Panc89 hA8 WT cells is shown in ( C ). ADAM8 can be detected as active and remnant ADAM8 in EVs. The negative control Calnexin was not detectable in isolated EVs. The measured activity of Panc89 hA8 WT- and KO-derived EVs on PepDAB #13 is displayed in ( D ) and is upregulated in Panc89 hA8 WT-derived EVs ( n = 2). ( E ) Representative images of immunofluorescence staining of Panc89 hA8 rescue cells with Hoechst dye (upper left), TSG101 (green; upper right), and ADAM8 (red; lower left). Merged images are displayed in the lower right and show that TSG101 shows little or no co-localization with ADAM8. Scale bar, 50 μm. ( F ) shows the detection of ADAM8, Flotillin-1, and CD81 via Western blot of Panc89 hA8 WT CL and EV preparations isolated from Panc89 hA8 WT, Panc89 hA8 rescue, Panc89 hA8 ΔCD rescue, and Panc89 hA8 KO cells. ADAM8 is detectable in all EV preparations except in EVs isolated from hA8 KO cells. Data are presented as mean values ± S.D.

Techniques Used: Derivative Assay, Isolation, Electron Microscopy, Western Blot, Negative Control, Activity Assay, Immunofluorescence, Staining

13) Product Images from "CD9P-1 expression correlates with the metastatic status of lung cancer, and a truncated form of CD9P-1, GS-168AT2, inhibits in vivo tumour growth"

Article Title: CD9P-1 expression correlates with the metastatic status of lung cancer, and a truncated form of CD9P-1, GS-168AT2, inhibits in vivo tumour growth

Journal: British Journal of Cancer

doi: 10.1038/sj.bjc.6606033

The degradation of GS-168AT2 is associated with its interaction with CD9 and CD81. ( A ) NCI-H460 cells were incubated for 2 h with (lanes labelled with +) or without (lanes labelled with −) GS-168AT2, followed by lysing with native lysis buffer. Proteins were immunoprecipitated with 229T mAb (lanes labelled with IP 229T mAb), anti-CD9 antibody (lanes labelled with IP CD9), anti-CD81 antibody (lanes labelled with IP CD81), anti- β 1-integrin antibody (lanes labelled with IP integrin), or anti-vascular endothelial factor receptor (IP Flk 1) (lanes labelled with IP Flk1). The obtained immunoprecipitates or total cell lysates of cells incubated with GS-168AT2 (lane labelled with total cell lysate) (as control) were resolved in SDS–PAGE under reducing conditions and western blotted with 229T mAb. GS-168AT2 was undetectable with the immunoprecipitates obtained with the anti- β 1-integrin (IP β 1-integrin) antibody and with the antivascular endothelial cell growth factor receptor (IP Flk 1), but was detectable with the immunoprecipitates obtained with 229T mAb (IP 229T mAb). Representative image of the western blot of three independent experiments is presented. ( B ) The immunoprecipitates obtained with either 229T mAb (lanes labelled with IP 229T mAb) or anti-CD9 antibody (lanes labelled with IP CD9), in the presence or absence of GS-168AT2 as in panel A of this figure, were also resolved in SDS–PAGE and immunoblotted with anti-CD9 antibody. CD9 was well immunoprecipitated with anti-CD9 antibody under our experimental conditions, as described in panel A.
Figure Legend Snippet: The degradation of GS-168AT2 is associated with its interaction with CD9 and CD81. ( A ) NCI-H460 cells were incubated for 2 h with (lanes labelled with +) or without (lanes labelled with −) GS-168AT2, followed by lysing with native lysis buffer. Proteins were immunoprecipitated with 229T mAb (lanes labelled with IP 229T mAb), anti-CD9 antibody (lanes labelled with IP CD9), anti-CD81 antibody (lanes labelled with IP CD81), anti- β 1-integrin antibody (lanes labelled with IP integrin), or anti-vascular endothelial factor receptor (IP Flk 1) (lanes labelled with IP Flk1). The obtained immunoprecipitates or total cell lysates of cells incubated with GS-168AT2 (lane labelled with total cell lysate) (as control) were resolved in SDS–PAGE under reducing conditions and western blotted with 229T mAb. GS-168AT2 was undetectable with the immunoprecipitates obtained with the anti- β 1-integrin (IP β 1-integrin) antibody and with the antivascular endothelial cell growth factor receptor (IP Flk 1), but was detectable with the immunoprecipitates obtained with 229T mAb (IP 229T mAb). Representative image of the western blot of three independent experiments is presented. ( B ) The immunoprecipitates obtained with either 229T mAb (lanes labelled with IP 229T mAb) or anti-CD9 antibody (lanes labelled with IP CD9), in the presence or absence of GS-168AT2 as in panel A of this figure, were also resolved in SDS–PAGE and immunoblotted with anti-CD9 antibody. CD9 was well immunoprecipitated with anti-CD9 antibody under our experimental conditions, as described in panel A.

Techniques Used: Incubation, Lysis, Immunoprecipitation, SDS Page, Western Blot

GS-168AT2 inhibits the in vivo tumour growth associated with drastic in vivo downregulation of CD9 in the tumour core, but not in CD81. ( A ) Tumour masses were extracted from animal cores at the end of treatment, homogenised, resolved in SDS–PAGE, and immunoblotted with anti-CD81 antibody. Commercial control cell lysates for CD9 (Santa Cruz) were used as control. Lane labelled with vehicle: tumours from animals treated with vehicle; lane labelled with GS-168AT2: tumours from animals treated with GS-168AT2. ( B ) Tumour masses were extracted from animals at the end of treatments (either after five successive days of treatment, lanes labelled under 5 days of treatment, or after complete treatment, as described in Figure 6 and under Materials and Methods section), homogenised, resolved in SDS–PAGE, and immunoblotted with either anti-CD9 antibody (upper panel) or anti-GAPDH antibody (lower panel) as control. CD9 expression was then appreciated (middle panel) relative to the internal control (GAPDH). Results representative of two independent experiments are presented.
Figure Legend Snippet: GS-168AT2 inhibits the in vivo tumour growth associated with drastic in vivo downregulation of CD9 in the tumour core, but not in CD81. ( A ) Tumour masses were extracted from animal cores at the end of treatment, homogenised, resolved in SDS–PAGE, and immunoblotted with anti-CD81 antibody. Commercial control cell lysates for CD9 (Santa Cruz) were used as control. Lane labelled with vehicle: tumours from animals treated with vehicle; lane labelled with GS-168AT2: tumours from animals treated with GS-168AT2. ( B ) Tumour masses were extracted from animals at the end of treatments (either after five successive days of treatment, lanes labelled under 5 days of treatment, or after complete treatment, as described in Figure 6 and under Materials and Methods section), homogenised, resolved in SDS–PAGE, and immunoblotted with either anti-CD9 antibody (upper panel) or anti-GAPDH antibody (lower panel) as control. CD9 expression was then appreciated (middle panel) relative to the internal control (GAPDH). Results representative of two independent experiments are presented.

Techniques Used: In Vivo, SDS Page, Expressing

14) Product Images from "Plasmatic exosomes from prostate cancer patients show increased carbonic anhydrase IX expression and activity and low pH"

Article Title: Plasmatic exosomes from prostate cancer patients show increased carbonic anhydrase IX expression and activity and low pH

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

doi: 10.1080/14756366.2019.1697249

Western blot analysis of plasmatic exosomes from PCa patients and CTR after 30% sucrose density gradient ultracentrifugation for housekeeping markers (Alix and CD81) and CA IX expression. (A) Protein characterisation of exosomal fractions purified from plasma of PCa patients performed with anti-Alix, anti-CD81 and M75 (CA IX). (B) Protein characterisation of exosomal fractions purified from plasma of CTR performed with anti-Alix, anti-CD81 and M75 (CA IX).
Figure Legend Snippet: Western blot analysis of plasmatic exosomes from PCa patients and CTR after 30% sucrose density gradient ultracentrifugation for housekeeping markers (Alix and CD81) and CA IX expression. (A) Protein characterisation of exosomal fractions purified from plasma of PCa patients performed with anti-Alix, anti-CD81 and M75 (CA IX). (B) Protein characterisation of exosomal fractions purified from plasma of CTR performed with anti-Alix, anti-CD81 and M75 (CA IX).

Techniques Used: Western Blot, Expressing, Purification

Detection, quantification and characterisation of CAIX+/CD81+ exosomes purified from PCa and CTR plasma by ELISA test. ELISA analysis of CAIX and CD81 expression in exosomes purified from plasma of 8 PCa patients and 8 CTR (1 ml for each sample). Rabbit polyclonal anti-CD81 antibody was used for the capture of exosomes on the plate. Expression levels of exosomal CAIX were expressed as means ± ES. The p values was
Figure Legend Snippet: Detection, quantification and characterisation of CAIX+/CD81+ exosomes purified from PCa and CTR plasma by ELISA test. ELISA analysis of CAIX and CD81 expression in exosomes purified from plasma of 8 PCa patients and 8 CTR (1 ml for each sample). Rabbit polyclonal anti-CD81 antibody was used for the capture of exosomes on the plate. Expression levels of exosomal CAIX were expressed as means ± ES. The p values was

Techniques Used: Purification, Enzyme-linked Immunosorbent Assay, Expressing

Nanoscale flow cytometry of plasma exosomes in PCa and CTR for intraluminal pH evaluation. The cytometer was calibrated using a mixture of non-fluorescent silica beads and fluorescent (green) latex beads with sizes from 110 nm to 1300 nm. The exosome preparation derived from plasma of 8 PCa patients and 8 CTR were stained 20 min at RT with anti-CD81 antibody and BCECF AM (10 µM) and analysed using flow cytometry. The double-positive events were then analysed for their size, based on the calibration with beads. Cumulative data are shown of the absolute number of CD81+/BCECF + exosomes of size less than 180 nm recovered from the plasma samples. Data are expressed as means ± SE. The p values was
Figure Legend Snippet: Nanoscale flow cytometry of plasma exosomes in PCa and CTR for intraluminal pH evaluation. The cytometer was calibrated using a mixture of non-fluorescent silica beads and fluorescent (green) latex beads with sizes from 110 nm to 1300 nm. The exosome preparation derived from plasma of 8 PCa patients and 8 CTR were stained 20 min at RT with anti-CD81 antibody and BCECF AM (10 µM) and analysed using flow cytometry. The double-positive events were then analysed for their size, based on the calibration with beads. Cumulative data are shown of the absolute number of CD81+/BCECF + exosomes of size less than 180 nm recovered from the plasma samples. Data are expressed as means ± SE. The p values was

Techniques Used: Flow Cytometry, Cytometry, Derivative Assay, Staining

15) Product Images from "Hepatitis C virus enters liver cells using the CD81 receptor complex proteins calpain-5 and CBLB"

Article Title: Hepatitis C virus enters liver cells using the CD81 receptor complex proteins calpain-5 and CBLB

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007111

A subset of CD81 interacting proteins is required for full HCV infectivity. ( .
Figure Legend Snippet: A subset of CD81 interacting proteins is required for full HCV infectivity. ( .

Techniques Used: Infection

CAPN5 and CBLB are cytoplasmic proteins enriched in the CD81 complex. (A) Whole cell proteome quantification for Lunet N hCD81 cells. Expression level as iBAQ value indicated for the CD81 interactor CAPN5 (red) and the HCV entry factors CD81 (green), SCARB1 (black square), CLDN1 (black hexagon) and OCLN (black diamond). Albumin (black dot) shown as additional positive control. (B) Comparison of protein abundance in whole cell lysates and protein enrichment in CD81 co-IPs from Lunet N hCD81 cells. CAPN5 (red) and CD81 (green) are highlighted. Dotted lines indicate median values of all detected proteins. (C, D) Flow cytometric staining of CAPN5 and CBLB on the surface of naïve Lunet N hCD81 cells or after membrane permeabilization reveals intracellular localization of CAPN5 and CBLB (E) A subfraction of CAPN5 and CBLB colocalizes with the membrane marker ZO-1. Lunet N CRISPR scrambled cells were stained with anti-ZO-1 and anti-CAPN5 (upper panel) or anti-CBLB (lower panel). Nuclei were stained with DAPI. Arrowheads indicate colocalization of ZO-1 and CAPN5 or CBLB. Representative confocal images; scale bars 10 μm. (F) Pearson’s correlation coefficient for ZO-1 and CAPN5 or CBLB calculated by intensity correlation analysis. Each symbol represents an individual frame; horizontal lines indicate the mean ± SEM.
Figure Legend Snippet: CAPN5 and CBLB are cytoplasmic proteins enriched in the CD81 complex. (A) Whole cell proteome quantification for Lunet N hCD81 cells. Expression level as iBAQ value indicated for the CD81 interactor CAPN5 (red) and the HCV entry factors CD81 (green), SCARB1 (black square), CLDN1 (black hexagon) and OCLN (black diamond). Albumin (black dot) shown as additional positive control. (B) Comparison of protein abundance in whole cell lysates and protein enrichment in CD81 co-IPs from Lunet N hCD81 cells. CAPN5 (red) and CD81 (green) are highlighted. Dotted lines indicate median values of all detected proteins. (C, D) Flow cytometric staining of CAPN5 and CBLB on the surface of naïve Lunet N hCD81 cells or after membrane permeabilization reveals intracellular localization of CAPN5 and CBLB (E) A subfraction of CAPN5 and CBLB colocalizes with the membrane marker ZO-1. Lunet N CRISPR scrambled cells were stained with anti-ZO-1 and anti-CAPN5 (upper panel) or anti-CBLB (lower panel). Nuclei were stained with DAPI. Arrowheads indicate colocalization of ZO-1 and CAPN5 or CBLB. Representative confocal images; scale bars 10 μm. (F) Pearson’s correlation coefficient for ZO-1 and CAPN5 or CBLB calculated by intensity correlation analysis. Each symbol represents an individual frame; horizontal lines indicate the mean ± SEM.

Techniques Used: Expressing, Positive Control, Protein Enrichment, Flow Cytometry, Staining, Marker, CRISPR

Stratification of 33 CD81 receptor interactions in primary human hepatocytes. ( A) Schematic overview of the experimental setup used to define the CD81-interactome in primary human hepatocytes (PHH). (B) Immunoblot analysis of CD81- and IgG-IPs from PHH of two donors using an anti-CD81 antibody. Actin served as loading control. L = lysate, FT = flow through, E = eluate. (C) LFQ intensities of proteins in CD81- or IgG-IPs from PHH of two independent donors. CD81 (green) and SCARB1 (black) served as positive and APOL2 (white) as negative control. CAPN5 (red) was discovered as CD81 interactor in PHH. (D) Scatter plot comparing intensity differences of proteins found in CD81- versus IgG-IPs in two donors of PHH. CD81 (green), SCARB1 (black), APOL2 (white) and CAPN5 (red) are highlighted. (E) Number of proteins found ≥ 10-fold enriched in the indicated co-IPs and membrane associated protein fraction. (F) 23 proteins found at least 4-fold enriched in CD81-IPs from PHH donor 1 and 2 and significantly enriched in co-IPs from Lunet N hCD81 and Lunet N hCD81HA. (G) 26 proteins identified in CD81-IPs from PHH donor 1 and 2 and in Lunet N hCD81 cells with high stringency (FDR
Figure Legend Snippet: Stratification of 33 CD81 receptor interactions in primary human hepatocytes. ( A) Schematic overview of the experimental setup used to define the CD81-interactome in primary human hepatocytes (PHH). (B) Immunoblot analysis of CD81- and IgG-IPs from PHH of two donors using an anti-CD81 antibody. Actin served as loading control. L = lysate, FT = flow through, E = eluate. (C) LFQ intensities of proteins in CD81- or IgG-IPs from PHH of two independent donors. CD81 (green) and SCARB1 (black) served as positive and APOL2 (white) as negative control. CAPN5 (red) was discovered as CD81 interactor in PHH. (D) Scatter plot comparing intensity differences of proteins found in CD81- versus IgG-IPs in two donors of PHH. CD81 (green), SCARB1 (black), APOL2 (white) and CAPN5 (red) are highlighted. (E) Number of proteins found ≥ 10-fold enriched in the indicated co-IPs and membrane associated protein fraction. (F) 23 proteins found at least 4-fold enriched in CD81-IPs from PHH donor 1 and 2 and significantly enriched in co-IPs from Lunet N hCD81 and Lunet N hCD81HA. (G) 26 proteins identified in CD81-IPs from PHH donor 1 and 2 and in Lunet N hCD81 cells with high stringency (FDR

Techniques Used: Flow Cytometry, Negative Control

16) Product Images from "Ethanol Induces Extracellular Vesicle Secretion by Altering Lipid Metabolism through the Mitochondria-Associated ER Membranes and Sphingomyelinases"

Article Title: Ethanol Induces Extracellular Vesicle Secretion by Altering Lipid Metabolism through the Mitochondria-Associated ER Membranes and Sphingomyelinases

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms22168438

Ethanol activates BV2 microglial cells, increases EV release and alters EV inflammatory molecule concentration. ( A ) Immunoblot analysis and quantification (au, arbitrary units) of p-ERK, p-p38 and p-p65 in cell extracts of ethanol (50 mM)-treated BV2 cells for different time periods (0, 0.5, 1, 3, 7 and 24 h). Blots were stripped, and the total quantities of ERK, p38, p65 and GAPDH were also assessed. A representative immunoblot of each protein is shown. ( B ) The BV2 cells treated with or without ethanol for 24 h were exposed to fluorescein-labeled latex beads for 30 min to internalize them, which reveals the phagocytic activity of these cells. Cells were labeled with tomato lectin. Scale bar, 20 µm. A representative photomicrograph from three different experiments is shown. Graph bars represent the mean number of latex beads phagocytosed by cells for each condition. The xyz axes projections obtained using confocal microscopy (lower panel) show that latex beads are internalized in the cytoplasm. Green channel and blue channel represent cell membranes and nuclei staining, respectively. Figure S2A,C in the Supplementary Material shows the panoramic images and fluorescence composition. ( C ) Electron microscopy images of microglia-derived EVs. Scale bar, 100 nm. Figure S2B in the Supplementary Material shows the panoramic images. ( D ) Measurement of the absolute size range and concentration of the EVs derived from microglia by the nanoparticles tracking analysis. ( E , F ) Analysis of the levels of the exosome protein markers CD81 and HSP70 ( E ) and the inflammatory-related proteins TLR4, NLRP3 and IL-1R ( F ) present in microglia-derived EVs upon 50 mM ethanol stimulation. A representative immunoblot for each protein and their molecular weight are shown. In each lane, 30 μg of protein were loaded. CD81 was used as the loading control. ( G ) Bar graphs represent the expression of the following miRNAs; miR-146a-5p, miR-21-5p and let-7b; after ethanol treatment (or no treatment) in BV2 microglial cells. Data represent mean ± SEM, n = 5 independent experiments. * p
Figure Legend Snippet: Ethanol activates BV2 microglial cells, increases EV release and alters EV inflammatory molecule concentration. ( A ) Immunoblot analysis and quantification (au, arbitrary units) of p-ERK, p-p38 and p-p65 in cell extracts of ethanol (50 mM)-treated BV2 cells for different time periods (0, 0.5, 1, 3, 7 and 24 h). Blots were stripped, and the total quantities of ERK, p38, p65 and GAPDH were also assessed. A representative immunoblot of each protein is shown. ( B ) The BV2 cells treated with or without ethanol for 24 h were exposed to fluorescein-labeled latex beads for 30 min to internalize them, which reveals the phagocytic activity of these cells. Cells were labeled with tomato lectin. Scale bar, 20 µm. A representative photomicrograph from three different experiments is shown. Graph bars represent the mean number of latex beads phagocytosed by cells for each condition. The xyz axes projections obtained using confocal microscopy (lower panel) show that latex beads are internalized in the cytoplasm. Green channel and blue channel represent cell membranes and nuclei staining, respectively. Figure S2A,C in the Supplementary Material shows the panoramic images and fluorescence composition. ( C ) Electron microscopy images of microglia-derived EVs. Scale bar, 100 nm. Figure S2B in the Supplementary Material shows the panoramic images. ( D ) Measurement of the absolute size range and concentration of the EVs derived from microglia by the nanoparticles tracking analysis. ( E , F ) Analysis of the levels of the exosome protein markers CD81 and HSP70 ( E ) and the inflammatory-related proteins TLR4, NLRP3 and IL-1R ( F ) present in microglia-derived EVs upon 50 mM ethanol stimulation. A representative immunoblot for each protein and their molecular weight are shown. In each lane, 30 μg of protein were loaded. CD81 was used as the loading control. ( G ) Bar graphs represent the expression of the following miRNAs; miR-146a-5p, miR-21-5p and let-7b; after ethanol treatment (or no treatment) in BV2 microglial cells. Data represent mean ± SEM, n = 5 independent experiments. * p

Techniques Used: Concentration Assay, Labeling, Activity Assay, Confocal Microscopy, Staining, Fluorescence, Electron Microscopy, Derivative Assay, Molecular Weight, Expressing

17) Product Images from "Persistent hepatitis C virus infections and hepatopathological manifestations in immune-competent humanized mice"

Article Title: Persistent hepatitis C virus infections and hepatopathological manifestations in immune-competent humanized mice

Journal: Cell Research

doi: 10.1038/cr.2014.116

HCV persistent infection of C/O Tg mice. HCV RNA in serum and liver after WT and C/O Tg (A - B) , or C Tg and C/O Tg (H - I) mice were infected with HCV for the indicated time. (C , D) HCV RNA in serum (C) and liver (D) after ICR-C/O Tg mice were infected with the indicated dose of HCV J399EM for 2 weeks. (E - G) HCV RNA in serum and liver of C/O Tg mice after infection with Con1/JFH1 ( E , TCID50 = 5 × 10 6 /ml), or patient sera positive for HCV2a ( F , 1 × 10 5 IU) or HCV1b ( G , 1.4 × 10 5 IU). Each symbol in F - G indicates HCV RNA copies in individual C/O Tg mouse. (J) PHT Tg cells (1 × 10 6 ) were infected with HCV (MOI = 1) for 72 h, in the presence of blocking antibodies to CD81 (2 μg, sc70804, Santa Cruz), OCLN (2 μg, ab64482, Abcam) or isotype control, respectively. HCV RNA levels in cells (left) and supernatants (right) were determined. Unless indicated, 1 ml HCV J399EM (TCID50 = 1 × 10 8 /ml) was used and HCV copies were expressed in log scales. Three mice at each time point or different treatment were used unless indicated above the bar. All data are shown as mean ± SD of at least three independent experiments. #, not detectable. Dashed grey lines indicate limit of detection, 100 copies/mg for liver tissue detection and 500 copies/ml for serum detection.
Figure Legend Snippet: HCV persistent infection of C/O Tg mice. HCV RNA in serum and liver after WT and C/O Tg (A - B) , or C Tg and C/O Tg (H - I) mice were infected with HCV for the indicated time. (C , D) HCV RNA in serum (C) and liver (D) after ICR-C/O Tg mice were infected with the indicated dose of HCV J399EM for 2 weeks. (E - G) HCV RNA in serum and liver of C/O Tg mice after infection with Con1/JFH1 ( E , TCID50 = 5 × 10 6 /ml), or patient sera positive for HCV2a ( F , 1 × 10 5 IU) or HCV1b ( G , 1.4 × 10 5 IU). Each symbol in F - G indicates HCV RNA copies in individual C/O Tg mouse. (J) PHT Tg cells (1 × 10 6 ) were infected with HCV (MOI = 1) for 72 h, in the presence of blocking antibodies to CD81 (2 μg, sc70804, Santa Cruz), OCLN (2 μg, ab64482, Abcam) or isotype control, respectively. HCV RNA levels in cells (left) and supernatants (right) were determined. Unless indicated, 1 ml HCV J399EM (TCID50 = 1 × 10 8 /ml) was used and HCV copies were expressed in log scales. Three mice at each time point or different treatment were used unless indicated above the bar. All data are shown as mean ± SD of at least three independent experiments. #, not detectable. Dashed grey lines indicate limit of detection, 100 copies/mg for liver tissue detection and 500 copies/ml for serum detection.

Techniques Used: Infection, Mouse Assay, Blocking Assay

18) Product Images from "Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages"

Article Title: Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-9-9

Flow cytometry detection of surface molecules on exosomes from saliva, plasma and breast milk. Exosomes from saliva, plasma and breast milk captured on anti-MHC class II beads were immunostained by using monoclonal antibodies against the tetraspanins CD9, CD63 and CD81 and analysed by flow cytometry. The antibodies (open peaks) were compared with their appropriate isotype controls (filled peaks).
Figure Legend Snippet: Flow cytometry detection of surface molecules on exosomes from saliva, plasma and breast milk. Exosomes from saliva, plasma and breast milk captured on anti-MHC class II beads were immunostained by using monoclonal antibodies against the tetraspanins CD9, CD63 and CD81 and analysed by flow cytometry. The antibodies (open peaks) were compared with their appropriate isotype controls (filled peaks).

Techniques Used: Flow Cytometry, Cytometry

Characterisation of breast milk exosomes by Western blot. The exosomal proteins from breast milk exosomes were loaded onto a 10% acrylamide gel and transferred to a nitrocellulose membrane. The breast milk exosomes are positive for Hsc70 and CD81, but negative for the endoplasmic reticulum protein, calnexin. Macrophage protein (
Figure Legend Snippet: Characterisation of breast milk exosomes by Western blot. The exosomal proteins from breast milk exosomes were loaded onto a 10% acrylamide gel and transferred to a nitrocellulose membrane. The breast milk exosomes are positive for Hsc70 and CD81, but negative for the endoplasmic reticulum protein, calnexin. Macrophage protein ("Cells") was used as positive control.

Techniques Used: Western Blot, Acrylamide Gel Assay, Positive Control

19) Product Images from "Improved isolation strategies to increase the yield and purity of human urinary exosomes for biomarker discovery"

Article Title: Improved isolation strategies to increase the yield and purity of human urinary exosomes for biomarker discovery

Journal: Scientific Reports

doi: 10.1038/s41598-018-22142-x

Protein markers and RNA load of exosomes, purified using UC-SEC. ( A ) In a representative experiment, 20 μl of fractions 1, 4–19 and 45 were tested by Western blotting with anti-CD81 antibody. NTA was used to count 50–150 nm particles in all SEC fractions, and the particle content of each lane indicated. Uncropped images of 2 blots (9 samples each + Mw markers) are presented. ( B ) Particle and protein concentrations in 50 fractions of the UC-SEC. The data are the mean ± SEM of 3 independent experiments. ( C ) Exosomal markers CD9, CD63, CD81 and TSG101 were tested by Western blotting. Shown are the UC pellet subsequently used for SEC, fractions 11 and 26 of the SEC and a pool of all exosome-containing SEC fractions of a representative experiment. One membrane was cut and probed with anti-TSG101 + anti-CD81 (left) or anti-CD63 and anti-CD9 (right), and uncropped images combined. ( D ) Total RNA isolated from TEU-2 cells, total urine and urinary exosomes from UC-SEC was analysed by NanoChip. Length of detected RNA species is indicated (nt). ( E ) Pearson correlation between urine contents (chemical composition and total RNA) and uEV parameters (exosome miRNA read count, protein content and particle number). Only Pearson correlations with p-value ≤ 0.05 are shown (blue for positive correlation), the intensity of blue colour corresponding to the degree of correlation. The data are diagnostic values for urine composition and RNA, protein and exosome concentrations of 6 total urine samples, processed by the optimized UC-SEC method.
Figure Legend Snippet: Protein markers and RNA load of exosomes, purified using UC-SEC. ( A ) In a representative experiment, 20 μl of fractions 1, 4–19 and 45 were tested by Western blotting with anti-CD81 antibody. NTA was used to count 50–150 nm particles in all SEC fractions, and the particle content of each lane indicated. Uncropped images of 2 blots (9 samples each + Mw markers) are presented. ( B ) Particle and protein concentrations in 50 fractions of the UC-SEC. The data are the mean ± SEM of 3 independent experiments. ( C ) Exosomal markers CD9, CD63, CD81 and TSG101 were tested by Western blotting. Shown are the UC pellet subsequently used for SEC, fractions 11 and 26 of the SEC and a pool of all exosome-containing SEC fractions of a representative experiment. One membrane was cut and probed with anti-TSG101 + anti-CD81 (left) or anti-CD63 and anti-CD9 (right), and uncropped images combined. ( D ) Total RNA isolated from TEU-2 cells, total urine and urinary exosomes from UC-SEC was analysed by NanoChip. Length of detected RNA species is indicated (nt). ( E ) Pearson correlation between urine contents (chemical composition and total RNA) and uEV parameters (exosome miRNA read count, protein content and particle number). Only Pearson correlations with p-value ≤ 0.05 are shown (blue for positive correlation), the intensity of blue colour corresponding to the degree of correlation. The data are diagnostic values for urine composition and RNA, protein and exosome concentrations of 6 total urine samples, processed by the optimized UC-SEC method.

Techniques Used: Purification, Size-exclusion Chromatography, Western Blot, Isolation, Diagnostic Assay

20) Product Images from "Characterization and Small RNA Content of Extracellular Vesicles in Follicular Fluid of Developing Bovine Antral Follicles"

Article Title: Characterization and Small RNA Content of Extracellular Vesicles in Follicular Fluid of Developing Bovine Antral Follicles

Journal: Scientific Reports

doi: 10.1038/srep25486

Quantitative and qualitative analysis of the EVs from small, medium and large follicles. ( A ) Western blot analysis of extracellular vesicle (CD81 and Alix), endoplasmic reticulum (GP96) and cellular (actin) markers in EV and cellular lysates. Equal amounts of total protein (10 μg) were loaded into each lane. ( B ) Representative CD81 protein in sucrose gradient fractions of small, medium and large follicles. Each fraction density was determined by refractometry and an equal volume of the resulting resuspended pellets was loaded for western blot analysis (n = 3). Input represents an aliquot of the EV sample that was applied to the sucrose gradient. ( C ) Representative transmission electron microscopy image of a thin section through an EV pellet isolated from a small follicle–several vesicles diameters (nm) are labeled.
Figure Legend Snippet: Quantitative and qualitative analysis of the EVs from small, medium and large follicles. ( A ) Western blot analysis of extracellular vesicle (CD81 and Alix), endoplasmic reticulum (GP96) and cellular (actin) markers in EV and cellular lysates. Equal amounts of total protein (10 μg) were loaded into each lane. ( B ) Representative CD81 protein in sucrose gradient fractions of small, medium and large follicles. Each fraction density was determined by refractometry and an equal volume of the resulting resuspended pellets was loaded for western blot analysis (n = 3). Input represents an aliquot of the EV sample that was applied to the sucrose gradient. ( C ) Representative transmission electron microscopy image of a thin section through an EV pellet isolated from a small follicle–several vesicles diameters (nm) are labeled.

Techniques Used: Western Blot, Transmission Assay, Electron Microscopy, Isolation, Labeling

21) Product Images from "Phenotype and Response to PAMPs of Human Monocyte-Derived Foam Cells Obtained by Long-Term Culture in the Presence of oxLDLs"

Article Title: Phenotype and Response to PAMPs of Human Monocyte-Derived Foam Cells Obtained by Long-Term Culture in the Presence of oxLDLs

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2020.01592

Western blot analysis of CD11b, CD36, CD47, and CD81 in prolonged- hMDMs and hMDFCs. Whole-cell lysates (25 μg) from prolonged- hMDMs and hMDFCs were analyzed using immunoblotting. Enhanced chemiluminescence signal was detected with Bio-Rad ChemiDoc XRS+ system. β-actin level was determined as a loading control. (A) Images shown are representative of four independent experiments (donors). (B) Quantification of protein levels. The optical density was measured for the bands of interest and normalized to β-actin signal. To compare expression levels of receptors, the relative optical density was calculated (prolonged-hMDFCs vs. corresponding prolonged-hMDMs). Results represents the mean ± SD from four independent experiments. Due to semi-quantitative nature of measurements, statistical analysis was not performed.
Figure Legend Snippet: Western blot analysis of CD11b, CD36, CD47, and CD81 in prolonged- hMDMs and hMDFCs. Whole-cell lysates (25 μg) from prolonged- hMDMs and hMDFCs were analyzed using immunoblotting. Enhanced chemiluminescence signal was detected with Bio-Rad ChemiDoc XRS+ system. β-actin level was determined as a loading control. (A) Images shown are representative of four independent experiments (donors). (B) Quantification of protein levels. The optical density was measured for the bands of interest and normalized to β-actin signal. To compare expression levels of receptors, the relative optical density was calculated (prolonged-hMDFCs vs. corresponding prolonged-hMDMs). Results represents the mean ± SD from four independent experiments. Due to semi-quantitative nature of measurements, statistical analysis was not performed.

Techniques Used: Western Blot, Expressing

22) Product Images from "Exosomes of Epstein-Barr Virus-Associated Gastric Carcinoma Suppress Dendritic Cell Maturation"

Article Title: Exosomes of Epstein-Barr Virus-Associated Gastric Carcinoma Suppress Dendritic Cell Maturation

Journal: Microorganisms

doi: 10.3390/microorganisms8111776

Detection of exosome marker proteins in gastric cancer cell lines with and without EBV-infection (MKN7, MKN7 + EBV, MKN74, and MKN74 + EBV) and the gastric cancer cell line of EBVaGC (SNU719). ( A ): PCR evaluation of an EBV-latent gene, EBNA1 and RT-PCR evaluation of a lytic gene, BZLF1 . ( B , C ): Western blot analysis of exosome marker proteins in cell lysates ( B ) and exosomes ( C ). Proteins from the same amount of cell lysate (20 μg of protein per each sample) or exosomal preparations from the same number of cells from gastric cancer cell lines (equivalent to the amount of exosome in 5 × 10 5 cells per each sample) were separated by SDS-PAGE, transferred onto a PVDF membrane, and incubated with specific antibodies (CD63, CD81, β-Actin). Signals were detected with HRP-conjugated secondary antibody and by using a chemiluminescence kit.
Figure Legend Snippet: Detection of exosome marker proteins in gastric cancer cell lines with and without EBV-infection (MKN7, MKN7 + EBV, MKN74, and MKN74 + EBV) and the gastric cancer cell line of EBVaGC (SNU719). ( A ): PCR evaluation of an EBV-latent gene, EBNA1 and RT-PCR evaluation of a lytic gene, BZLF1 . ( B , C ): Western blot analysis of exosome marker proteins in cell lysates ( B ) and exosomes ( C ). Proteins from the same amount of cell lysate (20 μg of protein per each sample) or exosomal preparations from the same number of cells from gastric cancer cell lines (equivalent to the amount of exosome in 5 × 10 5 cells per each sample) were separated by SDS-PAGE, transferred onto a PVDF membrane, and incubated with specific antibodies (CD63, CD81, β-Actin). Signals were detected with HRP-conjugated secondary antibody and by using a chemiluminescence kit.

Techniques Used: Marker, Infection, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot, SDS Page, Incubation

23) Product Images from "Mesenchymal Stem Cell-Derived Extracellular Vesicles Protect Human Corneal Endothelial Cells from Endoplasmic Reticulum Stress-Mediated Apoptosis"

Article Title: Mesenchymal Stem Cell-Derived Extracellular Vesicles Protect Human Corneal Endothelial Cells from Endoplasmic Reticulum Stress-Mediated Apoptosis

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms22094930

Characterization of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) and blood serum-derived EVs (SER-EVs). ( A ) Representative nanoparticle tracking analysis showing the EV size distribution. ( B ) Super resolution microscopy micrographs showing the pattern distribution of CD63 in green, CD81 in red, and CD9 in blue for one MSC-EV and SER-EV. Scale bar: 50 nm. ( C ) Legend showing the 39 antibodies used in the assay and their respective colors in the dot plots. ( D ) MACSPlex representative dot plots showing the MSC-EV and SER-EV distribution of allophycocyanin (APC)-stained bead populations; captured EVs are counterstained with APC-labeled detection antibodies using a mixture of anti-CD9, anti-CD63, and anti-CD81 (pan tetraspanins) antibodies. ( E ) Representative quantification of the median APC fluorescence positive values for the bead populations after background correction, clustered in different graphs according to their classification: tetraspanins, immunological, mesenchymal, and endothelial markers.
Figure Legend Snippet: Characterization of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) and blood serum-derived EVs (SER-EVs). ( A ) Representative nanoparticle tracking analysis showing the EV size distribution. ( B ) Super resolution microscopy micrographs showing the pattern distribution of CD63 in green, CD81 in red, and CD9 in blue for one MSC-EV and SER-EV. Scale bar: 50 nm. ( C ) Legend showing the 39 antibodies used in the assay and their respective colors in the dot plots. ( D ) MACSPlex representative dot plots showing the MSC-EV and SER-EV distribution of allophycocyanin (APC)-stained bead populations; captured EVs are counterstained with APC-labeled detection antibodies using a mixture of anti-CD9, anti-CD63, and anti-CD81 (pan tetraspanins) antibodies. ( E ) Representative quantification of the median APC fluorescence positive values for the bead populations after background correction, clustered in different graphs according to their classification: tetraspanins, immunological, mesenchymal, and endothelial markers.

Techniques Used: Derivative Assay, Microscopy, Staining, Labeling, Fluorescence

24) Product Images from "TGF-β1 promoted chondrocyte proliferation by regulating Sp1 through MSC-exosomes derived miR-135b"

Article Title: TGF-β1 promoted chondrocyte proliferation by regulating Sp1 through MSC-exosomes derived miR-135b

Journal: Cell Cycle

doi: 10.1080/15384101.2018.1556063

MiRNAs expression in MSC-exosomes treated with TGF-β1. MSC-exosomes were isolated from MSCs or MSCs that stimulated by 10 ng/ml TGF-β1 (TGF-β1-exosome). (a) Morphology of exosomes observed by transmission electron microscopy (TEM). Scale bar: 100 nm. (b) Exosome surface markers (CD63, CD81 and CD9) measured using western blot. Control group is culture medium. (c) The expression of the surface markers of MSC-exosomes. (d) MiRNAs expression in MSC-exosomes. * P
Figure Legend Snippet: MiRNAs expression in MSC-exosomes treated with TGF-β1. MSC-exosomes were isolated from MSCs or MSCs that stimulated by 10 ng/ml TGF-β1 (TGF-β1-exosome). (a) Morphology of exosomes observed by transmission electron microscopy (TEM). Scale bar: 100 nm. (b) Exosome surface markers (CD63, CD81 and CD9) measured using western blot. Control group is culture medium. (c) The expression of the surface markers of MSC-exosomes. (d) MiRNAs expression in MSC-exosomes. * P

Techniques Used: Expressing, Isolation, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Western Blot

25) Product Images from "Membrane-Associated RING-CH Proteins Associate with Bap31 and Target CD81 and CD44 to Lysosomes"

Article Title: Membrane-Associated RING-CH Proteins Associate with Bap31 and Target CD81 and CD44 to Lysosomes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0015132

CD81 and CD44 are targeted to lysosomes by MARCH proteins. Flag-tagged MARCH-constructs were transfected into HeLa cells and the cells were treated with 25 mM NH 4 Cl for 2 h. Cells were fixed and stained with anti-Flag and anti-CD81 or anti-CD44 antibodies (A) or stained with either CD81 or anti-CD44 antibodies along with rabbit anti-Lamp1 (B) as described in Materials and Methods . Mouse antibodies were detected with anti-mouse Alexa 488 (green) and rabbit antibodies with anti-rabbit Alexa 594 (red). In B, MARCH-expressing cells, inferred by the altered distribution of CD81 and CD44, are indicated with an asterisk (*). Bar, 10 µm (B).
Figure Legend Snippet: CD81 and CD44 are targeted to lysosomes by MARCH proteins. Flag-tagged MARCH-constructs were transfected into HeLa cells and the cells were treated with 25 mM NH 4 Cl for 2 h. Cells were fixed and stained with anti-Flag and anti-CD81 or anti-CD44 antibodies (A) or stained with either CD81 or anti-CD44 antibodies along with rabbit anti-Lamp1 (B) as described in Materials and Methods . Mouse antibodies were detected with anti-mouse Alexa 488 (green) and rabbit antibodies with anti-rabbit Alexa 594 (red). In B, MARCH-expressing cells, inferred by the altered distribution of CD81 and CD44, are indicated with an asterisk (*). Bar, 10 µm (B).

Techniques Used: Construct, Transfection, Staining, Expressing

Depletion of MARCH-IV affects surface expression of CD81. (A): MARCH expression in HFF was analyzed using real-time PCR and the PCR products were separated on a 1% agarose gel and visualized using sybr green (left panel). MARCH mRNA expression is shown as the fold change between samples containing HFF cDNA and no template control samples (right panel). (B): HFFs were treated with siRNA against MARCH-I, -IV, or –VIII as indicated. Cells were treated with siRNA four times over the course of 7 days. The success of each siRNA treatment was determined by monitoring the reduction of MARCH mRNA levels using real time quantitative PCR. (C): In parallel, cells were harvested via trypsinization and the surface levels of CD44 and CD81 were measured using flow cytometry. Graphs are displayed as percent mean florescence intensity.
Figure Legend Snippet: Depletion of MARCH-IV affects surface expression of CD81. (A): MARCH expression in HFF was analyzed using real-time PCR and the PCR products were separated on a 1% agarose gel and visualized using sybr green (left panel). MARCH mRNA expression is shown as the fold change between samples containing HFF cDNA and no template control samples (right panel). (B): HFFs were treated with siRNA against MARCH-I, -IV, or –VIII as indicated. Cells were treated with siRNA four times over the course of 7 days. The success of each siRNA treatment was determined by monitoring the reduction of MARCH mRNA levels using real time quantitative PCR. (C): In parallel, cells were harvested via trypsinization and the surface levels of CD44 and CD81 were measured using flow cytometry. Graphs are displayed as percent mean florescence intensity.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, SYBR Green Assay, Flow Cytometry, Cytometry

Downregulation of CD44 and CD81 by other members of the MARCH-family. HeLa cells were transfected with plasmids expressing cDNAs for each MARCH protein and the surface expression of CD81 and CD44 were assayed by flow cytometry (A) or confocal immune fluorescence analysis (B and C).
Figure Legend Snippet: Downregulation of CD44 and CD81 by other members of the MARCH-family. HeLa cells were transfected with plasmids expressing cDNAs for each MARCH protein and the surface expression of CD81 and CD44 were assayed by flow cytometry (A) or confocal immune fluorescence analysis (B and C).

Techniques Used: Transfection, Expressing, Flow Cytometry, Cytometry, Fluorescence

CD44 and CD81 are differentially expressed following MARCH-VIII expression. (A): To confirm that CD44 and CD81 were removed from the cell surface, HFF's were infected as above. 24 hours post-infection cells were harvested and processed for flow cytometry. Samples transduced with Ad-MARCH-VIII (line) display reduced surface expression of CD44 and CD81 compared to samples infected with Ad-Tet alone (fill). Expression of MARCH-VIII was confirmed by surface staining for the known MARCH-VIII substrate TfR. (B): HFFs were transduced with Ad-WT, Ad-K5, Ad-Vpu, or Ad-MARCH-VIII. 24 hours post-transduction cells were harvested and whole cell lysates were analyzed for the abundance of CD44 and CD81 using immunoblot. Samples infected with Ad-MARCH-VIII display significantly reduced levels of both CD44 and CD81 compared to either Mock infected samples or samples infected with other control adenoviruses. Equal protein loading was confirmed by immunoblotting for the ER resident chaperone Bap31 as well as ponceau red staining.
Figure Legend Snippet: CD44 and CD81 are differentially expressed following MARCH-VIII expression. (A): To confirm that CD44 and CD81 were removed from the cell surface, HFF's were infected as above. 24 hours post-infection cells were harvested and processed for flow cytometry. Samples transduced with Ad-MARCH-VIII (line) display reduced surface expression of CD44 and CD81 compared to samples infected with Ad-Tet alone (fill). Expression of MARCH-VIII was confirmed by surface staining for the known MARCH-VIII substrate TfR. (B): HFFs were transduced with Ad-WT, Ad-K5, Ad-Vpu, or Ad-MARCH-VIII. 24 hours post-transduction cells were harvested and whole cell lysates were analyzed for the abundance of CD44 and CD81 using immunoblot. Samples infected with Ad-MARCH-VIII display significantly reduced levels of both CD44 and CD81 compared to either Mock infected samples or samples infected with other control adenoviruses. Equal protein loading was confirmed by immunoblotting for the ER resident chaperone Bap31 as well as ponceau red staining.

Techniques Used: Expressing, Infection, Flow Cytometry, Cytometry, Transduction, Staining

26) Product Images from "Binding of the Hepatitis C Virus E2 Glycoprotein to CD81 Is Strain Specific and Is Modulated by a Complex Interplay between Hypervariable Regions 1 and 2"

Article Title: Binding of the Hepatitis C Virus E2 Glycoprotein to CD81 Is Strain Specific and Is Modulated by a Complex Interplay between Hypervariable Regions 1 and 2

Journal: Journal of Virology

doi: 10.1128/JVI.77.3.1856-1867.2003

Both the HVR1 and HVR2 regions of the N2 strain are required for maximal inhibition of E2-CD81 interaction, and mutation of residues 613 to 618 eliminates CD81 recognition. Complete E2 ectodomains and HVR-mutated E2 were tested for binding to bacterially expressed CD81 (GST-hCD81) by ELISA or to Molt-4 cells by FACS. (A) Reactivities of the E2 H complete ectodomain and the HVR1 N2 E2 H , HVR2 N2 E2 H , HVR1 N2 HVR2 N2 E2 H , and E2 H smut613-618 mutants. (B) Binding of the E2 N2 complete ectodomain and the HVR1 H HVR2 H E2 N2 and ΔHVR1HVR2 H E2 N2 mutants. Binding values are reported as the percentage of reactivity of the complete ectodomain and were calculated from dose-response curves of normalized amounts of the monomeric species of each protein, performed in duplicate.
Figure Legend Snippet: Both the HVR1 and HVR2 regions of the N2 strain are required for maximal inhibition of E2-CD81 interaction, and mutation of residues 613 to 618 eliminates CD81 recognition. Complete E2 ectodomains and HVR-mutated E2 were tested for binding to bacterially expressed CD81 (GST-hCD81) by ELISA or to Molt-4 cells by FACS. (A) Reactivities of the E2 H complete ectodomain and the HVR1 N2 E2 H , HVR2 N2 E2 H , HVR1 N2 HVR2 N2 E2 H , and E2 H smut613-618 mutants. (B) Binding of the E2 N2 complete ectodomain and the HVR1 H HVR2 H E2 N2 and ΔHVR1HVR2 H E2 N2 mutants. Binding values are reported as the percentage of reactivity of the complete ectodomain and were calculated from dose-response curves of normalized amounts of the monomeric species of each protein, performed in duplicate.

Techniques Used: Inhibition, Mutagenesis, Binding Assay, Enzyme-linked Immunosorbent Assay, FACS

Deletion of the HVR1 region improves efficiency of binding of E2 to CD81. Secreted recombinant proteins expressed by plasmids encoding the complete E2 ectodomains (black bars) or E2 with HVR1 deleted (white bars) from the H strain (A) or N2 strain (B) were tested for their ability to bind to bacterially expressed CD81 (GST-hCD81) by ELISA or to Molt-4 cells by FACS. Binding values are reported as the percentage of reactivity of the complete ectodomain and were calculated from dose-response curves of normalized amounts of the monomeric species of each protein, performed in duplicate.
Figure Legend Snippet: Deletion of the HVR1 region improves efficiency of binding of E2 to CD81. Secreted recombinant proteins expressed by plasmids encoding the complete E2 ectodomains (black bars) or E2 with HVR1 deleted (white bars) from the H strain (A) or N2 strain (B) were tested for their ability to bind to bacterially expressed CD81 (GST-hCD81) by ELISA or to Molt-4 cells by FACS. Binding values are reported as the percentage of reactivity of the complete ectodomain and were calculated from dose-response curves of normalized amounts of the monomeric species of each protein, performed in duplicate.

Techniques Used: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, FACS

E2 binding to CD81 is strain specific. (A) Western blot analysis of serial dilutions of the secreted E2 proteins expressed from plasmids encoding E2 from different viral isolates (E2 H , E2 BK , E2 N2 , and E2 J ). In nonreducing SDS-PAGE, E2 aggregates are the slow-migrating species, while the faster-migrating band corresponds to the monomer and is heterogeneous in size due to glycosylation. MW, molecular mass markers. (B) Secreted E2 proteins were normalized for their content in monomeric form and tested in a dose-response FACS-based experiment for binding to Molt 4 cells. ΔMFI represents the MFI with the background fluorescence subtracted. The data show the results of a representative experiment performed in triplicate. (C) FACS data, plotted as histograms of fluorescence intensity against relative cell number, for the binding on Molt-4 cells of a normalized dose of E2 monomer from each preparation of E2 variants. E2 H (dotted line), E2 N2 (thin line), E2 BK (dashed line), and E2 J (thick line) histograms are indicated by arrows; the grey histogram represents the fluorescence intensity measured with a supernatant from mock-transfected cells. (D) Binding values for the different E2 variants are reported as percentage of E2 H reactivity calculated as described in Materials and Methods. Binding to the bacterially expressed CD81 (GST-hCD81) was measured by ELISA, and the background signal observed on GST carrier protein was subtracted. Average values from two replicates were determined. Binding to Molt-4 cells was measured by FACS. The background signal measured in control reactions using equal amounts of cell culture supernatant from mock-transfected cells was subtracted from the MFI for each reaction.
Figure Legend Snippet: E2 binding to CD81 is strain specific. (A) Western blot analysis of serial dilutions of the secreted E2 proteins expressed from plasmids encoding E2 from different viral isolates (E2 H , E2 BK , E2 N2 , and E2 J ). In nonreducing SDS-PAGE, E2 aggregates are the slow-migrating species, while the faster-migrating band corresponds to the monomer and is heterogeneous in size due to glycosylation. MW, molecular mass markers. (B) Secreted E2 proteins were normalized for their content in monomeric form and tested in a dose-response FACS-based experiment for binding to Molt 4 cells. ΔMFI represents the MFI with the background fluorescence subtracted. The data show the results of a representative experiment performed in triplicate. (C) FACS data, plotted as histograms of fluorescence intensity against relative cell number, for the binding on Molt-4 cells of a normalized dose of E2 monomer from each preparation of E2 variants. E2 H (dotted line), E2 N2 (thin line), E2 BK (dashed line), and E2 J (thick line) histograms are indicated by arrows; the grey histogram represents the fluorescence intensity measured with a supernatant from mock-transfected cells. (D) Binding values for the different E2 variants are reported as percentage of E2 H reactivity calculated as described in Materials and Methods. Binding to the bacterially expressed CD81 (GST-hCD81) was measured by ELISA, and the background signal observed on GST carrier protein was subtracted. Average values from two replicates were determined. Binding to Molt-4 cells was measured by FACS. The background signal measured in control reactions using equal amounts of cell culture supernatant from mock-transfected cells was subtracted from the MFI for each reaction.

Techniques Used: Binding Assay, Western Blot, SDS Page, FACS, Fluorescence, Transfection, Enzyme-linked Immunosorbent Assay, Cell Culture

(A) Only monomeric E2 binds to CD81 or hepatic cell lines. Secreted E2 H was used for binding and pull-down experiments with different cell lines. Cell-bound E2 H was fractionated by SDS-PAGE under nonreducing conditions, and Western blot detection with anti-E2 rat MAb (61-a) followed by anti-rat horseradish peroxidase conjugate was performed. Lane 1, E2 H from crude supernatant of transfected 293 cells (input); lanes 2, 3, and 4, E2 H recovered after binding to Molt-4, HuH7, and HepG2-R2 cells, respectively; lanes 5 and 6, E2 H recovered after pull-down with mock-transfected CHO and CD81-transfected CHO cells, respectively. Migration of monomeric (M) and aggregated (A) E2 H species is indicated by arrows. Migration of molecular mass markers (MW) is shown on the left. (B) Soluble CD81 displaces E2 H binding to Molt-4 cells. The extracellular domain of CD81 fused to the GST protein competes the binding of E2 H to Molt-4 cells in a dose-dependent manner, while the GST protein alone has no inhibitory effect. (C) Temperature dependence of E2 H binding to Molt-4 cells. (D) E2-specific binding to the extracellular domain of bacterially expressed hCD81. Binding of E2 H to bacterially expressed human (GST-hCD81) and mouse (GST-mCD81) CD81 LEL fused to GST was measured by ELISA. One microgram of purified recombinant proteins was applied to the surfaces of microwell plates. Equal amounts of purified GST carrier protein were used as a negative control. Average values from two replicates are shown.
Figure Legend Snippet: (A) Only monomeric E2 binds to CD81 or hepatic cell lines. Secreted E2 H was used for binding and pull-down experiments with different cell lines. Cell-bound E2 H was fractionated by SDS-PAGE under nonreducing conditions, and Western blot detection with anti-E2 rat MAb (61-a) followed by anti-rat horseradish peroxidase conjugate was performed. Lane 1, E2 H from crude supernatant of transfected 293 cells (input); lanes 2, 3, and 4, E2 H recovered after binding to Molt-4, HuH7, and HepG2-R2 cells, respectively; lanes 5 and 6, E2 H recovered after pull-down with mock-transfected CHO and CD81-transfected CHO cells, respectively. Migration of monomeric (M) and aggregated (A) E2 H species is indicated by arrows. Migration of molecular mass markers (MW) is shown on the left. (B) Soluble CD81 displaces E2 H binding to Molt-4 cells. The extracellular domain of CD81 fused to the GST protein competes the binding of E2 H to Molt-4 cells in a dose-dependent manner, while the GST protein alone has no inhibitory effect. (C) Temperature dependence of E2 H binding to Molt-4 cells. (D) E2-specific binding to the extracellular domain of bacterially expressed hCD81. Binding of E2 H to bacterially expressed human (GST-hCD81) and mouse (GST-mCD81) CD81 LEL fused to GST was measured by ELISA. One microgram of purified recombinant proteins was applied to the surfaces of microwell plates. Equal amounts of purified GST carrier protein were used as a negative control. Average values from two replicates are shown.

Techniques Used: Binding Assay, SDS Page, Western Blot, Transfection, Migration, Enzyme-linked Immunosorbent Assay, Purification, Recombinant, Negative Control

E2 can bind to hepatic cells in a CD81-independent manner. (A) Binding to HepG2-R2 cells of the complete E2 H ectodomain and of mutant E2 H mut613-618 in the absence of competing antibodies or in the presence of the anti-CD81 MAb 1.3.3.22 or an unrelated MAb. (B) Binding of E2 H to Molt-4 or HepG2-R2 cells in the absence of competing antibodies or in the presence of the anti-CD81 MAb 1.3.3.22 or an unrelated MAb. Binding values are reported as the percentage of E2 H reactivity and were calculated from dose-response curves of normalized amounts of the monomeric species of each protein, performed in duplicate.
Figure Legend Snippet: E2 can bind to hepatic cells in a CD81-independent manner. (A) Binding to HepG2-R2 cells of the complete E2 H ectodomain and of mutant E2 H mut613-618 in the absence of competing antibodies or in the presence of the anti-CD81 MAb 1.3.3.22 or an unrelated MAb. (B) Binding of E2 H to Molt-4 or HepG2-R2 cells in the absence of competing antibodies or in the presence of the anti-CD81 MAb 1.3.3.22 or an unrelated MAb. Binding values are reported as the percentage of E2 H reactivity and were calculated from dose-response curves of normalized amounts of the monomeric species of each protein, performed in duplicate.

Techniques Used: Binding Assay, Mutagenesis

Human hepatic cells recognize E2 variants from different viral isolates with comparable efficiency. (A and E) FACS histograms of binding on HuH7 cells (A) and HepG2-R2 cells (E) of a normalized dose of E2 monomer from each preparation of E2 variants. E2 H (dotted lines), E2 N2 (thin lines) E2 BK (dashed lines), and E2 J (thick lines) histograms are indicated by arrows; the grey histograms represent the fluorescence intensity measured with a supernatant from mock-transfected cells. (B) Binding efficiencies of E2 proteins from different viral isolates (E2 H , E2 BK , E2 N2 , and E2 J ) on HuH7 cells or HepG2-R2 cells. Binding values are reported as the percentage of reactivity of the E2 H protein and were calculated from dose-response curves of normalized amounts of the monomeric species of each protein, performed in duplicate. (C) Cell surface expression of CD81 on the different cell lines measured by FACS analysis with an FITC-conjugated anti-CD81 MAb 1.3.3.22 (Santa Cruz) in a direct binding assay. (D) FACS histograms of the binding of the E2 H protein on HepG2 (thin line) and HepG2-R2 (thick line) cells. The grey histogram represents the fluorescence intensity measured using a supernatant from mock-transfected cells.
Figure Legend Snippet: Human hepatic cells recognize E2 variants from different viral isolates with comparable efficiency. (A and E) FACS histograms of binding on HuH7 cells (A) and HepG2-R2 cells (E) of a normalized dose of E2 monomer from each preparation of E2 variants. E2 H (dotted lines), E2 N2 (thin lines) E2 BK (dashed lines), and E2 J (thick lines) histograms are indicated by arrows; the grey histograms represent the fluorescence intensity measured with a supernatant from mock-transfected cells. (B) Binding efficiencies of E2 proteins from different viral isolates (E2 H , E2 BK , E2 N2 , and E2 J ) on HuH7 cells or HepG2-R2 cells. Binding values are reported as the percentage of reactivity of the E2 H protein and were calculated from dose-response curves of normalized amounts of the monomeric species of each protein, performed in duplicate. (C) Cell surface expression of CD81 on the different cell lines measured by FACS analysis with an FITC-conjugated anti-CD81 MAb 1.3.3.22 (Santa Cruz) in a direct binding assay. (D) FACS histograms of the binding of the E2 H protein on HepG2 (thin line) and HepG2-R2 (thick line) cells. The grey histogram represents the fluorescence intensity measured using a supernatant from mock-transfected cells.

Techniques Used: FACS, Binding Assay, Fluorescence, Transfection, Expressing

27) Product Images from "Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes"

Article Title: Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes

Journal: American Journal of Respiratory Cell and Molecular Biology

doi: 10.1165/rcmb.2015-0108OC

PE localizes to exosomes in airway epithelial cells and generates the neutrophil chemoattractant tripeptide Pro-Gly-Pro. ( A ) CFBE WT cells underwent immunofluorescence for PE ( top panels ) and classic exosomal markers of CD63, CD81, and Alix ( middle panels
Figure Legend Snippet: PE localizes to exosomes in airway epithelial cells and generates the neutrophil chemoattractant tripeptide Pro-Gly-Pro. ( A ) CFBE WT cells underwent immunofluorescence for PE ( top panels ) and classic exosomal markers of CD63, CD81, and Alix ( middle panels

Techniques Used: Immunofluorescence

28) Product Images from "Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs"

Article Title: Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs

Journal: Nature Communications

doi: 10.1038/ncomms3980

HnRNPA2B1 binds EXOmiRNAs through EXOmotifs and is involved in their loading into exosomes. ( a ) FACS analysis of hnRNPA2B1 and CD81 in exosome-coupled beads. Exosomes were coupled to aldehyde-sulfate beads, permeabilized or left intact and incubated with antibodies to hnRNPA2B1 (middle panels) or CD81 (right panels) and secondary antibody. Exosome-coupled beads incubated with secondary antibody alone were used as negative controls (left panels). ( b ) qPCR analysis of miRNAs contained in hnRNPA2B1 immunoprecipitates from exosome lysates, showing the specific binding of miR-198 to hnRNPA2B1 in exosomes. Immunoprecipitation was performed with magnetic beads coated with anti-hnRNPA2B1 or anti-IgG1 control antibody. Data are presented relative to miR-17 content in control immunoprecipitates. Error bars represent s.d. ( n =2). ( c ) Electrophoresis mobility shift assay showing the specific binding of miR-198 to hnRNPA2B1. Biotinylated miR-17, miR-198 or poly-A were incubated with or without purified human hnRNPA2B1 as indicated. ( d ) Electrophoresis mobility shift assay showing the binding of hnRNPA2B1 to wild-type and mutated miR-198. Biotinylated wild-type and mutated miR-198 were incubated with or without purified human hnRNPA2B1 as indicated. Numbers represent protein concentration (ng μl −1 ). ( e ) Electrophoresis mobility shift assay showing the binding of hnRNPA2B1 to miR-601. ( f ) qPCR analysis of miR-18a and miR-198 in exosomes from control cells or cells silenced with siRNAs against hnRNPA2B1. Bars represent miRNA levels in exosomes (arbitrary units). Error bars represent s.d. ( n =3). Student’s t -test; * P -value
Figure Legend Snippet: HnRNPA2B1 binds EXOmiRNAs through EXOmotifs and is involved in their loading into exosomes. ( a ) FACS analysis of hnRNPA2B1 and CD81 in exosome-coupled beads. Exosomes were coupled to aldehyde-sulfate beads, permeabilized or left intact and incubated with antibodies to hnRNPA2B1 (middle panels) or CD81 (right panels) and secondary antibody. Exosome-coupled beads incubated with secondary antibody alone were used as negative controls (left panels). ( b ) qPCR analysis of miRNAs contained in hnRNPA2B1 immunoprecipitates from exosome lysates, showing the specific binding of miR-198 to hnRNPA2B1 in exosomes. Immunoprecipitation was performed with magnetic beads coated with anti-hnRNPA2B1 or anti-IgG1 control antibody. Data are presented relative to miR-17 content in control immunoprecipitates. Error bars represent s.d. ( n =2). ( c ) Electrophoresis mobility shift assay showing the specific binding of miR-198 to hnRNPA2B1. Biotinylated miR-17, miR-198 or poly-A were incubated with or without purified human hnRNPA2B1 as indicated. ( d ) Electrophoresis mobility shift assay showing the binding of hnRNPA2B1 to wild-type and mutated miR-198. Biotinylated wild-type and mutated miR-198 were incubated with or without purified human hnRNPA2B1 as indicated. Numbers represent protein concentration (ng μl −1 ). ( e ) Electrophoresis mobility shift assay showing the binding of hnRNPA2B1 to miR-601. ( f ) qPCR analysis of miR-18a and miR-198 in exosomes from control cells or cells silenced with siRNAs against hnRNPA2B1. Bars represent miRNA levels in exosomes (arbitrary units). Error bars represent s.d. ( n =3). Student’s t -test; * P -value

Techniques Used: FACS, Incubation, Real-time Polymerase Chain Reaction, Binding Assay, Immunoprecipitation, Magnetic Beads, Electrophoresis, Mobility Shift, Purification, Protein Concentration

29) Product Images from "Binding of Hepatitis C Virus E2 Glycoprotein to CD81 Does Not Correlate with Species Permissiveness to Infection"

Article Title: Binding of Hepatitis C Virus E2 Glycoprotein to CD81 Does Not Correlate with Species Permissiveness to Infection

Journal: Journal of Virology

doi:

(A) Binding of E2 protein to recombinant, mutated CD81 molecules. ELISA plates were coated with the CD81 recombinant molecules, and 1:3 serial dilutions of crude lysate containing E2 were tested for binding. (B) Competition of E2 binding to t-CD81. t-CD81 was used to coat an ELISA plate. Competition was performed by preincubation of E2 extract with serial dilutions of recombinant CD81 molecules. ●, t-CD81; ○, t-CD81-F186L; ■, agm-CD81; □, agm-CD81-L186F.
Figure Legend Snippet: (A) Binding of E2 protein to recombinant, mutated CD81 molecules. ELISA plates were coated with the CD81 recombinant molecules, and 1:3 serial dilutions of crude lysate containing E2 were tested for binding. (B) Competition of E2 binding to t-CD81. t-CD81 was used to coat an ELISA plate. Competition was performed by preincubation of E2 extract with serial dilutions of recombinant CD81 molecules. ●, t-CD81; ○, t-CD81-F186L; ■, agm-CD81; □, agm-CD81-L186F.

Techniques Used: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay

(A) Binding of E2 protein to recombinant CD81 molecules. ELISA plates were coated with the CD81 recombinant molecules, and 1:3 serial dilutions of crude lysate containing E2 were tested for binding. (B) Competition of E2 binding to t-CD81. t-CD81 was used to coat an ELISA plate. Competition was performed by preincubation of E2 extract with serial dilutions of recombinant CD81 molecules. ●, t-CD81; ⧫, h-CD81; ■, agm-CD81; ▵, GST (control).
Figure Legend Snippet: (A) Binding of E2 protein to recombinant CD81 molecules. ELISA plates were coated with the CD81 recombinant molecules, and 1:3 serial dilutions of crude lysate containing E2 were tested for binding. (B) Competition of E2 binding to t-CD81. t-CD81 was used to coat an ELISA plate. Competition was performed by preincubation of E2 extract with serial dilutions of recombinant CD81 molecules. ●, t-CD81; ⧫, h-CD81; ■, agm-CD81; ▵, GST (control).

Techniques Used: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay

FACS analysis of E2 binding to B95-8 cell surface in the presence of recombinant t-CD81. Competition was performed by preincubation of subsaturating amounts of E2 with increasing concentrations of t-CD81-GST (●) or GST (▵). The median F.I. for binding of E2 in the absence of the competitor was 38.8, and the median F.I. for binding of the mock antigen was 2.8.
Figure Legend Snippet: FACS analysis of E2 binding to B95-8 cell surface in the presence of recombinant t-CD81. Competition was performed by preincubation of subsaturating amounts of E2 with increasing concentrations of t-CD81-GST (●) or GST (▵). The median F.I. for binding of E2 in the absence of the competitor was 38.8, and the median F.I. for binding of the mock antigen was 2.8.

Techniques Used: FACS, Binding Assay, Recombinant

FACS analysis of inhibition of E2 binding to B95-8 cells by anti-CD81 MAb. B95-8 cells were incubated with the monoclonal antibody at a concentration known to saturate the cell surface before the incubation with E2. Gray curve, E2 binding (median F.I., 34.2); open curve, binding competition by anti-CD81 MAb (median F.I., 5.3). The median F.I. for binding of the mock antigen to B95-8 was 2.7.
Figure Legend Snippet: FACS analysis of inhibition of E2 binding to B95-8 cells by anti-CD81 MAb. B95-8 cells were incubated with the monoclonal antibody at a concentration known to saturate the cell surface before the incubation with E2. Gray curve, E2 binding (median F.I., 34.2); open curve, binding competition by anti-CD81 MAb (median F.I., 5.3). The median F.I. for binding of the mock antigen to B95-8 was 2.7.

Techniques Used: FACS, Inhibition, Binding Assay, Incubation, Concentration Assay

30) Product Images from "Hepatitis C virus enters liver cells using the CD81 receptor complex proteins calpain-5 and CBLB"

Article Title: Hepatitis C virus enters liver cells using the CD81 receptor complex proteins calpain-5 and CBLB

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007111

Quantitative proteomics identifies 30 CD81 receptor interactions in hepatoma cells. (A) Schematic overview of the experimental setup used to define the CD81-interactome in human hepatoma cell lines. (B) Immunoblot analysis of CD81- and HA-IPs from Lunet N hCD81HA and Lunet N hCD81 cells using anti-CD81 or anti-HA antibodies, as indicated. GAPDH served as loading control. L = lysate, FT = flow through, E = eluate. Representative of 4 biological replicates. (C) LFQ intensities of proteins in CD81- and HA-IPs from the indicated cell line. CD81 (green) and SCARB1 (black) served as positive and APOL2 (white) as negative control. CAPN5 (red) was discovered as CD81 interactor in hepatoma cells. Median of 4 biological replicates. (D) Number of proteins significantly enriched in the indicated co-IPs and membrane associated fraction. (E) Volcano plot visualizing two-sample t-test comparing LFQ intensities of proteins found in CD81-IPs from Lunet N hCD81 and Lunet N. For each protein the t-test difference (log 10 ) of CD81 versus control co-IP of 4 biological replicates is plotted against the p value (-log 10 ). FDR = 0.01; s0 = 2. Proteins significantly enriched are highlighted in dark grey. CD81 (green), SCARB1 (black) APOL2 (white) and CAPN5 (red) are highlighted. (F) Overlap of significantly enriched proteins found in anti-CD81co-IPs from Lunet N hCD81 and Lunet N hCD81HA. See also S1 Fig , S2 Fig , S1 Table and S2 Table .
Figure Legend Snippet: Quantitative proteomics identifies 30 CD81 receptor interactions in hepatoma cells. (A) Schematic overview of the experimental setup used to define the CD81-interactome in human hepatoma cell lines. (B) Immunoblot analysis of CD81- and HA-IPs from Lunet N hCD81HA and Lunet N hCD81 cells using anti-CD81 or anti-HA antibodies, as indicated. GAPDH served as loading control. L = lysate, FT = flow through, E = eluate. Representative of 4 biological replicates. (C) LFQ intensities of proteins in CD81- and HA-IPs from the indicated cell line. CD81 (green) and SCARB1 (black) served as positive and APOL2 (white) as negative control. CAPN5 (red) was discovered as CD81 interactor in hepatoma cells. Median of 4 biological replicates. (D) Number of proteins significantly enriched in the indicated co-IPs and membrane associated fraction. (E) Volcano plot visualizing two-sample t-test comparing LFQ intensities of proteins found in CD81-IPs from Lunet N hCD81 and Lunet N. For each protein the t-test difference (log 10 ) of CD81 versus control co-IP of 4 biological replicates is plotted against the p value (-log 10 ). FDR = 0.01; s0 = 2. Proteins significantly enriched are highlighted in dark grey. CD81 (green), SCARB1 (black) APOL2 (white) and CAPN5 (red) are highlighted. (F) Overlap of significantly enriched proteins found in anti-CD81co-IPs from Lunet N hCD81 and Lunet N hCD81HA. See also S1 Fig , S2 Fig , S1 Table and S2 Table .

Techniques Used: Flow Cytometry, Negative Control, Co-Immunoprecipitation Assay

A subset of CD81 interacting proteins is required for full HCV infectivity. ( A) Functional map of host factors interacting with the HCV receptor CD81. Functional clusters (boxes) and previously reported interactions (bold lines) of the identified CD81 binding partners and the HCV entry factors OCLN and CLDN1 are depicted. Yellow lines between genes of different clusters indicate high-confidence ( > 0.9) STRING interactions. Lower confidence ( > 0.35) STRING interactions are shown as red lines. Nine highest scoring additional nodes (indicated by asterisk) were included for follow up analysis. The full set of identified proteins is depicted in S3A Fig . (B) Experimental setup of the siRNA screen used to identify CD81 interactors important for HCV infection. (C) Human hepatoma cells were transfected with a pool of three siRNAs targeting the 42 CD81-interactors or with a scrambled non-targeting control (SiSel NC), followed by infection with a HCV luciferase reporter virus (JcR-2A). Infectivity was measured 48 hpi as luciferase activity and normalized for cell viability and plate effects. Knock down of four CD81-interactors significantly decreased HCV infection (p≤ 0.05; abs (z score) ≥ 2). Data from 3 biological replicates shown as mean +SEM. See also S3 Fig .
Figure Legend Snippet: A subset of CD81 interacting proteins is required for full HCV infectivity. ( A) Functional map of host factors interacting with the HCV receptor CD81. Functional clusters (boxes) and previously reported interactions (bold lines) of the identified CD81 binding partners and the HCV entry factors OCLN and CLDN1 are depicted. Yellow lines between genes of different clusters indicate high-confidence ( > 0.9) STRING interactions. Lower confidence ( > 0.35) STRING interactions are shown as red lines. Nine highest scoring additional nodes (indicated by asterisk) were included for follow up analysis. The full set of identified proteins is depicted in S3A Fig . (B) Experimental setup of the siRNA screen used to identify CD81 interactors important for HCV infection. (C) Human hepatoma cells were transfected with a pool of three siRNAs targeting the 42 CD81-interactors or with a scrambled non-targeting control (SiSel NC), followed by infection with a HCV luciferase reporter virus (JcR-2A). Infectivity was measured 48 hpi as luciferase activity and normalized for cell viability and plate effects. Knock down of four CD81-interactors significantly decreased HCV infection (p≤ 0.05; abs (z score) ≥ 2). Data from 3 biological replicates shown as mean +SEM. See also S3 Fig .

Techniques Used: Infection, Functional Assay, Binding Assay, Transfection, Luciferase, Activity Assay

Plasmodium sporozoites use CD81, but not CAPN5 or CBLB for hepatoma cell entry. (A) Lunet N hCD81 human hepatoma cells were infected with sporozoites of a P . yoelii GFP reporter strain for 180 min, then fixed at 48 hpi, stained for the parasite protein UIS4 and the nuclear stain Hoechst 33342 and analyzed by fluorescence microscopy. Development of exoerythrocytic forms indicated by co-localization of GFP with the parasitophorous vacuole marker UIS4. Scale bar: 50 μm. (B) Lunet N and Lunet N hCD81 cells were infected as in (A) and productively infected cells quantified by fluorescence microscopy. Mean and SEM of 4 biological replicates shown. (C) Schematic overview of the experimental setup used to analyze the role of CD81 interacting proteins in P . yoelii entry into Lunet N hepatoma cells. (D) Human hepatoma cells were transfected with a pool of three siRNAs as described in Fig 3C , followed by infection with sporozoites of a P . yoelii GFP reporter strain. Infectivity was measured 24 hpi as formation of exoerythrocytic forms by microscopy. Knock down of CD81 significantly decreased P . yoelii infection (p≤ 0.05; abs (z score) ≥ 2). Data from 2 biological replicates shown as mean +SEM. See also S4 and S7 Figs.
Figure Legend Snippet: Plasmodium sporozoites use CD81, but not CAPN5 or CBLB for hepatoma cell entry. (A) Lunet N hCD81 human hepatoma cells were infected with sporozoites of a P . yoelii GFP reporter strain for 180 min, then fixed at 48 hpi, stained for the parasite protein UIS4 and the nuclear stain Hoechst 33342 and analyzed by fluorescence microscopy. Development of exoerythrocytic forms indicated by co-localization of GFP with the parasitophorous vacuole marker UIS4. Scale bar: 50 μm. (B) Lunet N and Lunet N hCD81 cells were infected as in (A) and productively infected cells quantified by fluorescence microscopy. Mean and SEM of 4 biological replicates shown. (C) Schematic overview of the experimental setup used to analyze the role of CD81 interacting proteins in P . yoelii entry into Lunet N hepatoma cells. (D) Human hepatoma cells were transfected with a pool of three siRNAs as described in Fig 3C , followed by infection with sporozoites of a P . yoelii GFP reporter strain. Infectivity was measured 24 hpi as formation of exoerythrocytic forms by microscopy. Knock down of CD81 significantly decreased P . yoelii infection (p≤ 0.05; abs (z score) ≥ 2). Data from 2 biological replicates shown as mean +SEM. See also S4 and S7 Figs.

Techniques Used: Infection, Staining, Fluorescence, Microscopy, Marker, Transfection

CAPN5 and CBLB support a postbinding step during HCV lipoviroparticle entry. (A) Schematic overview of the experimental setup used to analyze different steps of the HCV life cycle in the CRISPR/Cas9 knockout cell lines. (B) Infection of CAPN5 (red) and CBLB (blue) knockout and parental cell lines with HCV genotype 2 reporter virus. 72 hpi infection rates were quantified as luciferase activity and normalized to infection rates in cells transduced with a non-targeting scrambled sgRNA. CD81 knockout cells served as positive control. Data from 3 independent experiments shown as mean +SEM. (C) Flow cytometric surface staining of CD81, SCARB1, CLDN1 and OCLN in cells knocked out for CAPN5 (red) or CBLB (blue). Parental cells (black) served as positive control. Isotype control stainings or stainings with secondary antibody only (white) as negative controls. (D) Entry of lentiviral particles pseudotyped with glycoproteins from HCV GT1a (strain H77) or GT1b (strain Con1). Infectivity normalized to particles without envelope protein (negative control), to particles with VSV-G envelope (positive control) and to infection of cells transduced with non-targeting scrambled sgRNA. (E) Quantification of HCV fusion activity at the plasma membrane. Cells were pretreated with concanamycin A to inhibit endosomal acidification, cold-bound with HCV luciferase reporter virus (JcR-2A; 4°C, 2 h), shifted to 37°C (1 h) and washed with a pH 5 buffer to induce artificial plasma membrane fusion. A pH 7 buffer wash served to determine the background infection rate. 48 hpi infection rate was quantified as luciferase activity. Inh: flunarizine; scr: scrambled sgRNA (F) Immunofluorescence staining of cell lines electroporated with a HCV subgenomic replicon RNA (JFH1) at 48 hpt. Green: NS5A. Blue: DAPI. 10x magnification. (G) Cell lines were electroporated with wildtype HCV subgenomic replicon RNA (JFH1) or a polymerase active site mutant JFH1-ΔGDD (dotted lines), both encoding a luciferase reporter. Replication quantified as luciferase activity at the indicated time point post electroporation. Results normalized to the 4 h time point to account for electroporation efficiency. Data from at least three independent experiments shown as representative results (C, F) or as mean ± SEM (B, D, E, G). Significance according to unpaired t-test (B, E) or to MANOVA (G) indicated by * (p≤ 0.05), ** (p≤ 0.01), *** (p≤ 0.001). See also S6 Fig .
Figure Legend Snippet: CAPN5 and CBLB support a postbinding step during HCV lipoviroparticle entry. (A) Schematic overview of the experimental setup used to analyze different steps of the HCV life cycle in the CRISPR/Cas9 knockout cell lines. (B) Infection of CAPN5 (red) and CBLB (blue) knockout and parental cell lines with HCV genotype 2 reporter virus. 72 hpi infection rates were quantified as luciferase activity and normalized to infection rates in cells transduced with a non-targeting scrambled sgRNA. CD81 knockout cells served as positive control. Data from 3 independent experiments shown as mean +SEM. (C) Flow cytometric surface staining of CD81, SCARB1, CLDN1 and OCLN in cells knocked out for CAPN5 (red) or CBLB (blue). Parental cells (black) served as positive control. Isotype control stainings or stainings with secondary antibody only (white) as negative controls. (D) Entry of lentiviral particles pseudotyped with glycoproteins from HCV GT1a (strain H77) or GT1b (strain Con1). Infectivity normalized to particles without envelope protein (negative control), to particles with VSV-G envelope (positive control) and to infection of cells transduced with non-targeting scrambled sgRNA. (E) Quantification of HCV fusion activity at the plasma membrane. Cells were pretreated with concanamycin A to inhibit endosomal acidification, cold-bound with HCV luciferase reporter virus (JcR-2A; 4°C, 2 h), shifted to 37°C (1 h) and washed with a pH 5 buffer to induce artificial plasma membrane fusion. A pH 7 buffer wash served to determine the background infection rate. 48 hpi infection rate was quantified as luciferase activity. Inh: flunarizine; scr: scrambled sgRNA (F) Immunofluorescence staining of cell lines electroporated with a HCV subgenomic replicon RNA (JFH1) at 48 hpt. Green: NS5A. Blue: DAPI. 10x magnification. (G) Cell lines were electroporated with wildtype HCV subgenomic replicon RNA (JFH1) or a polymerase active site mutant JFH1-ΔGDD (dotted lines), both encoding a luciferase reporter. Replication quantified as luciferase activity at the indicated time point post electroporation. Results normalized to the 4 h time point to account for electroporation efficiency. Data from at least three independent experiments shown as representative results (C, F) or as mean ± SEM (B, D, E, G). Significance according to unpaired t-test (B, E) or to MANOVA (G) indicated by * (p≤ 0.05), ** (p≤ 0.01), *** (p≤ 0.001). See also S6 Fig .

Techniques Used: CRISPR, Knock-Out, Infection, Luciferase, Activity Assay, Transduction, Positive Control, Flow Cytometry, Staining, Negative Control, Immunofluorescence, Mutagenesis, Electroporation

Stratification of 33 CD81 receptor interactions in primary human hepatocytes. ( A) Schematic overview of the experimental setup used to define the CD81-interactome in primary human hepatocytes (PHH). (B) Immunoblot analysis of CD81- and IgG-IPs from PHH of two donors using an anti-CD81 antibody. Actin served as loading control. L = lysate, FT = flow through, E = eluate. (C) LFQ intensities of proteins in CD81- or IgG-IPs from PHH of two independent donors. CD81 (green) and SCARB1 (black) served as positive and APOL2 (white) as negative control. CAPN5 (red) was discovered as CD81 interactor in PHH. (D) Scatter plot comparing intensity differences of proteins found in CD81- versus IgG-IPs in two donors of PHH. CD81 (green), SCARB1 (black), APOL2 (white) and CAPN5 (red) are highlighted. (E) Number of proteins found ≥ 10-fold enriched in the indicated co-IPs and membrane associated protein fraction. (F) 23 proteins found at least 4-fold enriched in CD81-IPs from PHH donor 1 and 2 and significantly enriched in co-IPs from Lunet N hCD81 and Lunet N hCD81HA. (G) 26 proteins identified in CD81-IPs from PHH donor 1 and 2 and in Lunet N hCD81 cells with high stringency (FDR
Figure Legend Snippet: Stratification of 33 CD81 receptor interactions in primary human hepatocytes. ( A) Schematic overview of the experimental setup used to define the CD81-interactome in primary human hepatocytes (PHH). (B) Immunoblot analysis of CD81- and IgG-IPs from PHH of two donors using an anti-CD81 antibody. Actin served as loading control. L = lysate, FT = flow through, E = eluate. (C) LFQ intensities of proteins in CD81- or IgG-IPs from PHH of two independent donors. CD81 (green) and SCARB1 (black) served as positive and APOL2 (white) as negative control. CAPN5 (red) was discovered as CD81 interactor in PHH. (D) Scatter plot comparing intensity differences of proteins found in CD81- versus IgG-IPs in two donors of PHH. CD81 (green), SCARB1 (black), APOL2 (white) and CAPN5 (red) are highlighted. (E) Number of proteins found ≥ 10-fold enriched in the indicated co-IPs and membrane associated protein fraction. (F) 23 proteins found at least 4-fold enriched in CD81-IPs from PHH donor 1 and 2 and significantly enriched in co-IPs from Lunet N hCD81 and Lunet N hCD81HA. (G) 26 proteins identified in CD81-IPs from PHH donor 1 and 2 and in Lunet N hCD81 cells with high stringency (FDR

Techniques Used: Flow Cytometry, Negative Control

CAPN5 and CBLB are cytoplasmic proteins enriched in the CD81 complex. (A) Whole cell proteome quantification for Lunet N hCD81 cells. Expression level as iBAQ value indicated for the CD81 interactor CAPN5 (red) and the HCV entry factors CD81 (green), SCARB1 (black square), CLDN1 (black hexagon) and OCLN (black diamond). Albumin (black dot) shown as additional positive control. (B) Comparison of protein abundance in whole cell lysates and protein enrichment in CD81 co-IPs from Lunet N hCD81 cells. CAPN5 (red) and CD81 (green) are highlighted. Dotted lines indicate median values of all detected proteins. (C, D) Flow cytometric staining of CAPN5 and CBLB on the surface of naïve Lunet N hCD81 cells or after membrane permeabilization reveals intracellular localization of CAPN5 and CBLB (E) A subfraction of CAPN5 and CBLB colocalizes with the membrane marker ZO-1. Lunet N CRISPR scrambled cells were stained with anti-ZO-1 and anti-CAPN5 (upper panel) or anti-CBLB (lower panel). Nuclei were stained with DAPI. Arrowheads indicate colocalization of ZO-1 and CAPN5 or CBLB. Representative confocal images; scale bars 10 μm. (F) Pearson’s correlation coefficient for ZO-1 and CAPN5 or CBLB calculated by intensity correlation analysis. Each symbol represents an individual frame; horizontal lines indicate the mean ± SEM.
Figure Legend Snippet: CAPN5 and CBLB are cytoplasmic proteins enriched in the CD81 complex. (A) Whole cell proteome quantification for Lunet N hCD81 cells. Expression level as iBAQ value indicated for the CD81 interactor CAPN5 (red) and the HCV entry factors CD81 (green), SCARB1 (black square), CLDN1 (black hexagon) and OCLN (black diamond). Albumin (black dot) shown as additional positive control. (B) Comparison of protein abundance in whole cell lysates and protein enrichment in CD81 co-IPs from Lunet N hCD81 cells. CAPN5 (red) and CD81 (green) are highlighted. Dotted lines indicate median values of all detected proteins. (C, D) Flow cytometric staining of CAPN5 and CBLB on the surface of naïve Lunet N hCD81 cells or after membrane permeabilization reveals intracellular localization of CAPN5 and CBLB (E) A subfraction of CAPN5 and CBLB colocalizes with the membrane marker ZO-1. Lunet N CRISPR scrambled cells were stained with anti-ZO-1 and anti-CAPN5 (upper panel) or anti-CBLB (lower panel). Nuclei were stained with DAPI. Arrowheads indicate colocalization of ZO-1 and CAPN5 or CBLB. Representative confocal images; scale bars 10 μm. (F) Pearson’s correlation coefficient for ZO-1 and CAPN5 or CBLB calculated by intensity correlation analysis. Each symbol represents an individual frame; horizontal lines indicate the mean ± SEM.

Techniques Used: Expressing, Positive Control, Protein Enrichment, Flow Cytometry, Staining, Marker, CRISPR

31) Product Images from "Mesenchymal stem cells and cell-derived extracellular vesicles protect hippocampal neurons from oxidative stress and synapse damage induced by amyloid-β oligomers"

Article Title: Mesenchymal stem cells and cell-derived extracellular vesicles protect hippocampal neurons from oxidative stress and synapse damage induced by amyloid-β oligomers

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M117.807180

Size distribution and immunological characterization of EVs released by MSCs. A–E , MSC-derived EVs were isolated (see “Experimental Procedures”) and mounted on grids for electron microscopy, negatively stained, and imaged by TEM. Smaller vesicles with diameters ranging between 30 and 200 nm ( A–C ) and larger vesicles with diameters ranging between 400 and 600 ( D–E ) nm could be visualized. Vesicles indicated by arrows in A are shown in higher magnification in the inset. F , frequency distribution of vesicle diameter derived from TEM analysis ( n = 104 vesicles measured). Most (∼65%) of the vesicles ranged between 30 and 200 nm in diameter. G , dot blot analysis showing that EVs are immunoreactive for exosome-associated tetraspanins CD81 and CD63. H , NanoSight NTA. Size distribution of MSC-derived EVs showed predominance of particles with diameters ranging between 50 and 200 nm. Lines in different colors correspond to quintuplicate analyses from the same EV preparation. Scale bars , 200 nm ( A , C , and D ); 100 nm ( A ( inset ) and B ); and 500 nm ( E ). I , analysis of EV particle size distribution from forward scatter ( FSC ) and side scatter ( SSC ) by flow cytometry. J , microvesicles identified as CD90.1-positive particles. K , overlay of fluorescence histograms (normalized by the respective mode values) in a non-labeled sample (background control, gray curve ) and a CD90.1-labeled sample ( red curve ), showing the presence of 10.1% CD90.1-positive microvesicles in the EV population.
Figure Legend Snippet: Size distribution and immunological characterization of EVs released by MSCs. A–E , MSC-derived EVs were isolated (see “Experimental Procedures”) and mounted on grids for electron microscopy, negatively stained, and imaged by TEM. Smaller vesicles with diameters ranging between 30 and 200 nm ( A–C ) and larger vesicles with diameters ranging between 400 and 600 ( D–E ) nm could be visualized. Vesicles indicated by arrows in A are shown in higher magnification in the inset. F , frequency distribution of vesicle diameter derived from TEM analysis ( n = 104 vesicles measured). Most (∼65%) of the vesicles ranged between 30 and 200 nm in diameter. G , dot blot analysis showing that EVs are immunoreactive for exosome-associated tetraspanins CD81 and CD63. H , NanoSight NTA. Size distribution of MSC-derived EVs showed predominance of particles with diameters ranging between 50 and 200 nm. Lines in different colors correspond to quintuplicate analyses from the same EV preparation. Scale bars , 200 nm ( A , C , and D ); 100 nm ( A ( inset ) and B ); and 500 nm ( E ). I , analysis of EV particle size distribution from forward scatter ( FSC ) and side scatter ( SSC ) by flow cytometry. J , microvesicles identified as CD90.1-positive particles. K , overlay of fluorescence histograms (normalized by the respective mode values) in a non-labeled sample (background control, gray curve ) and a CD90.1-labeled sample ( red curve ), showing the presence of 10.1% CD90.1-positive microvesicles in the EV population.

Techniques Used: Derivative Assay, Isolation, Electron Microscopy, Staining, Transmission Electron Microscopy, Dot Blot, Flow Cytometry, Cytometry, Fluorescence, Labeling

32) Product Images from "Reproducible and scalable purification of extracellular vesicles using combined bind-elute and size exclusion chromatography"

Article Title: Reproducible and scalable purification of extracellular vesicles using combined bind-elute and size exclusion chromatography

Journal: Scientific Reports

doi: 10.1038/s41598-017-10646-x

EV surface protein profile and EV uptake analysis by flow cytometry. ( A ) Signal intensity of respective bead populations normalized to the EV markers CD81/CD9/CD63. ( B ) NTA total particle count of scatter and fluorescent EVs isolated with UC and TFF/BE-SEC. ( C ) Mean fluorescence intensity normalized over the control (∆MFI) comparing the two isolation methods (n = 2). ( D ) Representative overlaid histograms of UC and TFF/BE-SEC isolated EVs uptake assay on recipient Huh7 cells, compared to untreated Huh7 cells (UT).
Figure Legend Snippet: EV surface protein profile and EV uptake analysis by flow cytometry. ( A ) Signal intensity of respective bead populations normalized to the EV markers CD81/CD9/CD63. ( B ) NTA total particle count of scatter and fluorescent EVs isolated with UC and TFF/BE-SEC. ( C ) Mean fluorescence intensity normalized over the control (∆MFI) comparing the two isolation methods (n = 2). ( D ) Representative overlaid histograms of UC and TFF/BE-SEC isolated EVs uptake assay on recipient Huh7 cells, compared to untreated Huh7 cells (UT).

Techniques Used: Flow Cytometry, Cytometry, Isolation, Size-exclusion Chromatography, Fluorescence

33) Product Images from "Human umbilical cord mesenchymal stromal cells-derived extracellular vesicles exert potent bone protective effects by CLEC11A-mediated regulation of bone metabolism"

Article Title: Human umbilical cord mesenchymal stromal cells-derived extracellular vesicles exert potent bone protective effects by CLEC11A-mediated regulation of bone metabolism

Journal: Theranostics

doi: 10.7150/thno.39238

Characterization of hucMSCs and hucMSC-EVs. (A) hucMSCs showed a spindle fibroblast-like morphology. Scale bar: 100 μm. (B) hucMSCs were capable of differentiating into osteoblasts, adipocytes or chondrocytes after osteogenic, adipogenic or chondrogenic medium induction, indicated by Alizarin Red S (ARS) staining, Oil Red O (ORO) staining and Alcian Blue staining. Scale bars: 100 μm (left); 50 μm (middle); 100 μm (right). (C) Flow cytometry analysis of the typical surface markers in hucMSCs. Blank curves: the isotype controls; solid gray curves: the test samples. (D) Morphology of hucMSC-EVs under transmission electron microscopy. Scale bar: 100 nm. (E) Size distribution of hucMSC-EVs calculated by dynamic light scattering analysis. (F) Detection of the EV surface markers (CD9, CD63, CD81 and TSG101) in hucMSC-EVs by Western Blot.
Figure Legend Snippet: Characterization of hucMSCs and hucMSC-EVs. (A) hucMSCs showed a spindle fibroblast-like morphology. Scale bar: 100 μm. (B) hucMSCs were capable of differentiating into osteoblasts, adipocytes or chondrocytes after osteogenic, adipogenic or chondrogenic medium induction, indicated by Alizarin Red S (ARS) staining, Oil Red O (ORO) staining and Alcian Blue staining. Scale bars: 100 μm (left); 50 μm (middle); 100 μm (right). (C) Flow cytometry analysis of the typical surface markers in hucMSCs. Blank curves: the isotype controls; solid gray curves: the test samples. (D) Morphology of hucMSC-EVs under transmission electron microscopy. Scale bar: 100 nm. (E) Size distribution of hucMSC-EVs calculated by dynamic light scattering analysis. (F) Detection of the EV surface markers (CD9, CD63, CD81 and TSG101) in hucMSC-EVs by Western Blot.

Techniques Used: Staining, Flow Cytometry, Transmission Assay, Electron Microscopy, Western Blot

34) Product Images from "Immunogenicity and functional characterization of Leishmania-derived hepatitis C virus envelope glycoprotein complex"

Article Title: Immunogenicity and functional characterization of Leishmania-derived hepatitis C virus envelope glycoprotein complex

Journal: Scientific Reports

doi: 10.1038/srep30627

A functional analysis of the fE1E2 and tE1E2 complex expressed in L. tarentolae . ( A ) The GST-CD81-LEL pull-down assay. L. tarentolae cell wild-type lysate (WT) and lysates containing the recombinant E1E2 complexes were placed on glutathione–agarose beads preadsorbed with CD81-LEL fused to GST. After 16 h of incubation, the beads were washed and suspended in the SDS-PAGE sample buffer. Western blotting was performed with anti-E2, anti-E1, and anti-CD81 antibodies diluted 1:1000. ( B ) Analysis of the conformational epitopes of the fE1E2 and tE1E2 complex expressed in L. tarentolae . GNA-ELISA was performed with MAbs in the concentration of 5 μg/ml. R04 - isotype of the control MAb to a cytomegalovirus-specific protein. Huh 7.5 cell lysate containing gt1a/2a HCV chimera proteins was used as the positive control (vE1E2). The background from wild-type cell lysates was subtracted from obtained results. The error bars represent the standard deviations of 2 replicate values.
Figure Legend Snippet: A functional analysis of the fE1E2 and tE1E2 complex expressed in L. tarentolae . ( A ) The GST-CD81-LEL pull-down assay. L. tarentolae cell wild-type lysate (WT) and lysates containing the recombinant E1E2 complexes were placed on glutathione–agarose beads preadsorbed with CD81-LEL fused to GST. After 16 h of incubation, the beads were washed and suspended in the SDS-PAGE sample buffer. Western blotting was performed with anti-E2, anti-E1, and anti-CD81 antibodies diluted 1:1000. ( B ) Analysis of the conformational epitopes of the fE1E2 and tE1E2 complex expressed in L. tarentolae . GNA-ELISA was performed with MAbs in the concentration of 5 μg/ml. R04 - isotype of the control MAb to a cytomegalovirus-specific protein. Huh 7.5 cell lysate containing gt1a/2a HCV chimera proteins was used as the positive control (vE1E2). The background from wild-type cell lysates was subtracted from obtained results. The error bars represent the standard deviations of 2 replicate values.

Techniques Used: Functional Assay, Pull Down Assay, Recombinant, Incubation, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Positive Control

35) Product Images from "HCV-associated exosomes promote myeloid-derived suppressor cell expansion via inhibiting miR-124 to regulate T follicular cell differentiation and function"

Article Title: HCV-associated exosomes promote myeloid-derived suppressor cell expansion via inhibiting miR-124 to regulate T follicular cell differentiation and function

Journal: Cell Discovery

doi: 10.1038/s41421-018-0052-z

Characterization of exosomes isolated from HCV patient plasma and HCV-infected cells. a Identification of CD63, CD81 and HSP90 expressions in exosomes isolated from the plasma of HCV patients and HS (with exoEasy Maxi Kit) by immunoblotting. b Evaluation of CD63 concentration in the plasma and purified exosomes isolated from the same subject, or in the supernatant and enriched exosomes with exoEasy Maxi Kit. The same amount of proteins were loaded in the immunoblotting. c Detection of CD81 expression in exosomes (purified by CD81-coated beads) by flow cytometric analysis. d Electron microscope observation of exosomes. Exosomes isolated from the supernatant of HCV +/− Huh7 cells by differential centrifugation were immuno-stained with or without anti-CD81 antibody, followed by secondary antibody labeled with 12 nm gold particles, then subjected to IEM observation. Arrowed structures represent exosomes without gold labeling (left panel) and exosomes associated with gold particles (right panel). Bars = 50 or 100 nm as indicated. e Detection of HCV RNA by RT-PCR in exosomes purified by sequential centrifugation from HCV +/− subjects (upper panel) and quantification of their copy numbers in exosomes isolated from 1 ml plasma of HCV-infected patients by real-time RT-PCR. f Detection of HCV RNA by real-time RT-PCR in exosomes isolated by the centrifugation method from culture supernatants of HCV +/− Huh7 cells. HCV RNA copy number, shown as IU/ml supernatant-derived exosomes, is shown in the upper panel. Repeated experiments by RT-PCR measuring HCV RNA in exosomes purified by CD81-coated beads from the culture supernatant of HCV-infected (Huh7R) and -uninfected (Huh7) hepatocytes are shown in the lower panel. g Detection of HCV Core protein in exosomes isolated from the plasma of HCV +/− subjects or from the supernatants of HCV +/− Huh7 cells by exoEasy Maxi Kit followed by CD81-coated beads purification methods. Lower panel show exosomes isolated from the supernatants of HCV +/− Huh7 cells by the sequential centrifugation method. Cell lysate from Huh7 and Huh7R hepatocytes served as negative and positive controls. h Repeated experiments by RT-PCR measuring HCV RNA in CD33 + myeloid cells incubated with exosomes isolated from the supernatants of HCV +/− Huh7 cells overnight
Figure Legend Snippet: Characterization of exosomes isolated from HCV patient plasma and HCV-infected cells. a Identification of CD63, CD81 and HSP90 expressions in exosomes isolated from the plasma of HCV patients and HS (with exoEasy Maxi Kit) by immunoblotting. b Evaluation of CD63 concentration in the plasma and purified exosomes isolated from the same subject, or in the supernatant and enriched exosomes with exoEasy Maxi Kit. The same amount of proteins were loaded in the immunoblotting. c Detection of CD81 expression in exosomes (purified by CD81-coated beads) by flow cytometric analysis. d Electron microscope observation of exosomes. Exosomes isolated from the supernatant of HCV +/− Huh7 cells by differential centrifugation were immuno-stained with or without anti-CD81 antibody, followed by secondary antibody labeled with 12 nm gold particles, then subjected to IEM observation. Arrowed structures represent exosomes without gold labeling (left panel) and exosomes associated with gold particles (right panel). Bars = 50 or 100 nm as indicated. e Detection of HCV RNA by RT-PCR in exosomes purified by sequential centrifugation from HCV +/− subjects (upper panel) and quantification of their copy numbers in exosomes isolated from 1 ml plasma of HCV-infected patients by real-time RT-PCR. f Detection of HCV RNA by real-time RT-PCR in exosomes isolated by the centrifugation method from culture supernatants of HCV +/− Huh7 cells. HCV RNA copy number, shown as IU/ml supernatant-derived exosomes, is shown in the upper panel. Repeated experiments by RT-PCR measuring HCV RNA in exosomes purified by CD81-coated beads from the culture supernatant of HCV-infected (Huh7R) and -uninfected (Huh7) hepatocytes are shown in the lower panel. g Detection of HCV Core protein in exosomes isolated from the plasma of HCV +/− subjects or from the supernatants of HCV +/− Huh7 cells by exoEasy Maxi Kit followed by CD81-coated beads purification methods. Lower panel show exosomes isolated from the supernatants of HCV +/− Huh7 cells by the sequential centrifugation method. Cell lysate from Huh7 and Huh7R hepatocytes served as negative and positive controls. h Repeated experiments by RT-PCR measuring HCV RNA in CD33 + myeloid cells incubated with exosomes isolated from the supernatants of HCV +/− Huh7 cells overnight

Techniques Used: Isolation, Infection, Concentration Assay, Purification, Expressing, Flow Cytometry, Microscopy, Centrifugation, Staining, Labeling, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Derivative Assay, Incubation

36) Product Images from "Imaging of extracellular vesicles derived from human bone marrow mesenchymal stem cells using fluorescent and magnetic labels"

Article Title: Imaging of extracellular vesicles derived from human bone marrow mesenchymal stem cells using fluorescent and magnetic labels

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S159404

The SR-SIM analysis of hBM-MSCs with intracellular structures visible inside the cells positively stained with lypophilic dyes PKH26 ( A – C ) or tagged with superparamagnetic iron nanoparticles conjugated with rhodamine (Molday ION) ( D and E ) (red). Notes: Coexpression of tetraspanins (exosome markers), such as CD9 ( A and D ), CD63 ( B and E ), and CD81 ( C and F ) (green), was demonstrated. Cell nuclei were stained with Hoechst (blue). Scale bar =50 μm. Abbreviations: hBM-MSCs, human bone marrow mesenchymal stem cells; SR-SIM, super-resolution structured illumination microscopy.
Figure Legend Snippet: The SR-SIM analysis of hBM-MSCs with intracellular structures visible inside the cells positively stained with lypophilic dyes PKH26 ( A – C ) or tagged with superparamagnetic iron nanoparticles conjugated with rhodamine (Molday ION) ( D and E ) (red). Notes: Coexpression of tetraspanins (exosome markers), such as CD9 ( A and D ), CD63 ( B and E ), and CD81 ( C and F ) (green), was demonstrated. Cell nuclei were stained with Hoechst (blue). Scale bar =50 μm. Abbreviations: hBM-MSCs, human bone marrow mesenchymal stem cells; SR-SIM, super-resolution structured illumination microscopy.

Techniques Used: Staining, Microscopy

The SR-SIM analysis of hBM-MSCs, 24 hours after their co-culture with EVs previously stained with different dyes. Notes: EVs labeled with PKH26 ( A – C ) or tagged with Molday ION ( D – F ) (red) taken up by hBM-MSCs are visible inside the cells. Coexpression of tetraspanins: CD9 ( A and D ), CD63 ( B and E ), and CD81 ( C and F ) (green) were demonstrated. Cell nuclei were stained with Hoechst (blue). Scale bar =20 μm. Abbreviations: EVs, extracellular vesicles; hBM-MSCs, human bone marrow mesenchymal stem cells; SR-SIM, super-resolution structured illumination microscopy.
Figure Legend Snippet: The SR-SIM analysis of hBM-MSCs, 24 hours after their co-culture with EVs previously stained with different dyes. Notes: EVs labeled with PKH26 ( A – C ) or tagged with Molday ION ( D – F ) (red) taken up by hBM-MSCs are visible inside the cells. Coexpression of tetraspanins: CD9 ( A and D ), CD63 ( B and E ), and CD81 ( C and F ) (green) were demonstrated. Cell nuclei were stained with Hoechst (blue). Scale bar =20 μm. Abbreviations: EVs, extracellular vesicles; hBM-MSCs, human bone marrow mesenchymal stem cells; SR-SIM, super-resolution structured illumination microscopy.

Techniques Used: Co-Culture Assay, Staining, Labeling, Microscopy

37) Product Images from "Exosomes isolation and identification from equine mesenchymal stem cells"

Article Title: Exosomes isolation and identification from equine mesenchymal stem cells

Journal: BMC Veterinary Research

doi: 10.1186/s12917-019-1789-9

Immunogold labeled exosomes with anti CD81 antibody. Scale bar = 250 nm
Figure Legend Snippet: Immunogold labeled exosomes with anti CD81 antibody. Scale bar = 250 nm

Techniques Used: Labeling

Immunofluorescence staining of equine adipose derived stem cells with CD9 ( b ), CD63 ( c ) and CD81 ( d ). Negative control with non-immune serum ( a ). Scale bar = 25 μm
Figure Legend Snippet: Immunofluorescence staining of equine adipose derived stem cells with CD9 ( b ), CD63 ( c ) and CD81 ( d ). Negative control with non-immune serum ( a ). Scale bar = 25 μm

Techniques Used: Immunofluorescence, Staining, Derivative Assay, Negative Control

38) Product Images from "Delivery of miR-424-5p via Extracellular Vesicles Promotes the Apoptosis of MDA-MB-231 TNBC Cells in the Tumor Microenvironment"

Article Title: Delivery of miR-424-5p via Extracellular Vesicles Promotes the Apoptosis of MDA-MB-231 TNBC Cells in the Tumor Microenvironment

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms22020844

Isolation and characterization of extracellular vesicles (EVs) derived from adipose tissue-mesenchymal stromal cells (AT-MSCs). ( A ) EVs were isolated from the conditioned medium of AT-MSCs following the multicentrifugation method. ( B ) A phase-contrast transmission electron micrograph of the isolated exosomes. Scale bar: 100 nm. ( C ) Protein from AT-MSC cell lysates and EVs was analyzed by Western blotting with antibodies against CD9, CD63, CD81, CD105 and GAPDH. ( D ) Yields of EVs derived from AT-MSCs. Protein amounts and particle numbers of harvested EVs were determined by the Bradford method and nanoparticle tracking analysis (NTA), respectively. ( E ) Particle size distributions of EVs-424 were measured by NTA and showed a peak at 83 nm. ( F ) Bioanalyzer electropherogram of RNA extracted from EVs-424. The x -axis indicates the length of the RNA in nucleotides (nt), and the y -axis indicates the fluorescence intensity in arbitrary units. The lowest peak at 25 nt indicates the lower size marker. FU, fluorescent units.
Figure Legend Snippet: Isolation and characterization of extracellular vesicles (EVs) derived from adipose tissue-mesenchymal stromal cells (AT-MSCs). ( A ) EVs were isolated from the conditioned medium of AT-MSCs following the multicentrifugation method. ( B ) A phase-contrast transmission electron micrograph of the isolated exosomes. Scale bar: 100 nm. ( C ) Protein from AT-MSC cell lysates and EVs was analyzed by Western blotting with antibodies against CD9, CD63, CD81, CD105 and GAPDH. ( D ) Yields of EVs derived from AT-MSCs. Protein amounts and particle numbers of harvested EVs were determined by the Bradford method and nanoparticle tracking analysis (NTA), respectively. ( E ) Particle size distributions of EVs-424 were measured by NTA and showed a peak at 83 nm. ( F ) Bioanalyzer electropherogram of RNA extracted from EVs-424. The x -axis indicates the length of the RNA in nucleotides (nt), and the y -axis indicates the fluorescence intensity in arbitrary units. The lowest peak at 25 nt indicates the lower size marker. FU, fluorescent units.

Techniques Used: Isolation, Derivative Assay, Transmission Assay, Western Blot, Fluorescence, Marker

39) Product Images from "Evidence of a Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE) Machinery in HIV-1 Assembly and Release *"

Article Title: Evidence of a Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE) Machinery in HIV-1 Assembly and Release *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.241521

NSF-DN induces the cytosolic localization of CD63 and CD81. A and B , HeLa cells were transfected with vectors expressing HA-tagged NSF-WT or NSF-DN. 24 h after transfection, cells were fixed, stained with anti-HA antibody along with anti-CD63 ( A ) or anti-CD81
Figure Legend Snippet: NSF-DN induces the cytosolic localization of CD63 and CD81. A and B , HeLa cells were transfected with vectors expressing HA-tagged NSF-WT or NSF-DN. 24 h after transfection, cells were fixed, stained with anti-HA antibody along with anti-CD63 ( A ) or anti-CD81

Techniques Used: Transfection, Expressing, Staining

40) Product Images from "The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus"

Article Title: The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus

Journal: The EMBO Journal

doi: 10.1093/emboj/cdf529

Fig. 1. ( A ) Histograms showing the E2 binding of different viral isolates to human cell lines as measured by FACS analysis. Values are expressed as a percentage of the H77-E2 isolate binding. Molt-4 cells are represented by the black histogram. The human hepatic cell line Huh7 is represented by the dark gray histogram and the HepG2 cells by the light gray histogram. ( B ) Cell surface expression of CD81 measured on the different cell lines by FACS analysis with the anti-CD81 (mAb1.3.3.22) antibody in a direct binding assay. Molt-4 cells are represented by the triangle, Huh7 by the circle and HepG2 by the square. On the y axis net median fluorescence intensity (MFI) values are reported.
Figure Legend Snippet: Fig. 1. ( A ) Histograms showing the E2 binding of different viral isolates to human cell lines as measured by FACS analysis. Values are expressed as a percentage of the H77-E2 isolate binding. Molt-4 cells are represented by the black histogram. The human hepatic cell line Huh7 is represented by the dark gray histogram and the HepG2 cells by the light gray histogram. ( B ) Cell surface expression of CD81 measured on the different cell lines by FACS analysis with the anti-CD81 (mAb1.3.3.22) antibody in a direct binding assay. Molt-4 cells are represented by the triangle, Huh7 by the circle and HepG2 by the square. On the y axis net median fluorescence intensity (MFI) values are reported.

Techniques Used: Binding Assay, FACS, Expressing, Fluorescence

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  • 88
    Santa Cruz Biotechnology mouse monoclonal antibody cd81
    Morphological and Molecular Characterization of Exosomes from human Blastocoel Fluid. ( A , B ) Scanning Electron Micrographs of extracellular vesicles isolated from Follicular Fluid (FF) and Blastocoel Fluid (BF) respectively. ( C ) Diameter distribution of exosomes from BFs. Gauss fit of the Feret’s diameter histogram measured on SEM microscopies show an average BF diameter of 75 ± 3 nm and a full width at half maximum (FWHM) of 38 ± 8 nm. ( D ) Transmission Electron Microscopy images of exosomes from BFs. ( E ) Transmission Electron Microscopy images of exosomes from BFs marked with <t>CD81.</t> ( F ) Nanoparticle Tracking Analysis (NTA) of BF extracellular vesicles. Extracellular vesicles from Follicular Fluid were used as reference control (inset). Diameters and concentration of vesicles are indicated in the table. ( G ) ELISA assay with the tetraspanin CD63 antibody of BF exosomes. Amount (µg) of CD63 protein and EV concentration (number of particles/100 μl) evaluated in BFs. Follicular Fluid (FF) samples were used as reference control. Results are expressed as mean ± SEM.
    Mouse Monoclonal Antibody Cd81, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Santa Cruz Biotechnology goat anti cd81
    Inhibition of intracranial tumors by hUCBSC in vivo . (A) Nude mice with pre-established intracranial human glioma tumors (U251 or 5310) were treated with hUCBSC by intracranial injection (2.5×10 5 ). Fourteen days after hUCBSC administration, the brains were harvested, sectioned, and stained with Hematoxylin and Eosin (n≥3). Inset pictures show higher magnification at scale bar = 100 µm. (B) Characterization of hUCBSC in tumor areas in nude mice brain sections: Fourteen days after hUCBSC administration, the brains were harvested, sectioned and immunoprobed with mesenchymal stem cell markers CD29 and <t>CD81</t> using Alexa flour-594 secondary antibody. (n ≥ 3). Scale bar = 100 µm. (C) Upregulation of PTEN in nude mice: Mice brain sections were immunoprobed with PTEN and CD81 using appropriate fluorescence-conjugated secondary antibodies. Secondary antibodies used for PTEN and CD81 were: goat anti-mouse Alexa flour-594 for PTEN and donkey anti-goat Alexa Fluor 488 for CD81, respectively. Scale bar = 100 µm. (D) Downregulation of XIAP in mice: Mice brain sections were probed with XIAP antibody by DAB immunohistochemistry and counterstained with DAPI. Scale bar = 100 µm. Inset pictures show DAPI. (n = > 3).
    Goat Anti Cd81, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Morphological and Molecular Characterization of Exosomes from human Blastocoel Fluid. ( A , B ) Scanning Electron Micrographs of extracellular vesicles isolated from Follicular Fluid (FF) and Blastocoel Fluid (BF) respectively. ( C ) Diameter distribution of exosomes from BFs. Gauss fit of the Feret’s diameter histogram measured on SEM microscopies show an average BF diameter of 75 ± 3 nm and a full width at half maximum (FWHM) of 38 ± 8 nm. ( D ) Transmission Electron Microscopy images of exosomes from BFs. ( E ) Transmission Electron Microscopy images of exosomes from BFs marked with CD81. ( F ) Nanoparticle Tracking Analysis (NTA) of BF extracellular vesicles. Extracellular vesicles from Follicular Fluid were used as reference control (inset). Diameters and concentration of vesicles are indicated in the table. ( G ) ELISA assay with the tetraspanin CD63 antibody of BF exosomes. Amount (µg) of CD63 protein and EV concentration (number of particles/100 μl) evaluated in BFs. Follicular Fluid (FF) samples were used as reference control. Results are expressed as mean ± SEM.

    Journal: Scientific Reports

    Article Title: Identification of extracellular vesicles and characterization of miRNA expression profiles in human blastocoel fluid

    doi: 10.1038/s41598-018-36452-7

    Figure Lengend Snippet: Morphological and Molecular Characterization of Exosomes from human Blastocoel Fluid. ( A , B ) Scanning Electron Micrographs of extracellular vesicles isolated from Follicular Fluid (FF) and Blastocoel Fluid (BF) respectively. ( C ) Diameter distribution of exosomes from BFs. Gauss fit of the Feret’s diameter histogram measured on SEM microscopies show an average BF diameter of 75 ± 3 nm and a full width at half maximum (FWHM) of 38 ± 8 nm. ( D ) Transmission Electron Microscopy images of exosomes from BFs. ( E ) Transmission Electron Microscopy images of exosomes from BFs marked with CD81. ( F ) Nanoparticle Tracking Analysis (NTA) of BF extracellular vesicles. Extracellular vesicles from Follicular Fluid were used as reference control (inset). Diameters and concentration of vesicles are indicated in the table. ( G ) ELISA assay with the tetraspanin CD63 antibody of BF exosomes. Amount (µg) of CD63 protein and EV concentration (number of particles/100 μl) evaluated in BFs. Follicular Fluid (FF) samples were used as reference control. Results are expressed as mean ± SEM.

    Article Snippet: Then the grids were incubated in blocking solution 5% BSA for 1.30 hr at room temperature, rinsed with PBS and incubated in a humid chamber overnight at 4 °C with a mouse monoclonal antibody CD81 (Santa Cruz Biotechnology, Heidelberg, Germany) in a dilution 1:50 with TBS/BSA.

    Techniques: Isolation, Transmission Assay, Electron Microscopy, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Inhibition of intracranial tumors by hUCBSC in vivo . (A) Nude mice with pre-established intracranial human glioma tumors (U251 or 5310) were treated with hUCBSC by intracranial injection (2.5×10 5 ). Fourteen days after hUCBSC administration, the brains were harvested, sectioned, and stained with Hematoxylin and Eosin (n≥3). Inset pictures show higher magnification at scale bar = 100 µm. (B) Characterization of hUCBSC in tumor areas in nude mice brain sections: Fourteen days after hUCBSC administration, the brains were harvested, sectioned and immunoprobed with mesenchymal stem cell markers CD29 and CD81 using Alexa flour-594 secondary antibody. (n ≥ 3). Scale bar = 100 µm. (C) Upregulation of PTEN in nude mice: Mice brain sections were immunoprobed with PTEN and CD81 using appropriate fluorescence-conjugated secondary antibodies. Secondary antibodies used for PTEN and CD81 were: goat anti-mouse Alexa flour-594 for PTEN and donkey anti-goat Alexa Fluor 488 for CD81, respectively. Scale bar = 100 µm. (D) Downregulation of XIAP in mice: Mice brain sections were probed with XIAP antibody by DAB immunohistochemistry and counterstained with DAPI. Scale bar = 100 µm. Inset pictures show DAPI. (n = > 3).

    Journal: PLoS ONE

    Article Title: Upregulation of PTEN in Glioma Cells by Cord Blood Mesenchymal Stem Cells Inhibits Migration via Downregulation of the PI3K/Akt Pathway

    doi: 10.1371/journal.pone.0010350

    Figure Lengend Snippet: Inhibition of intracranial tumors by hUCBSC in vivo . (A) Nude mice with pre-established intracranial human glioma tumors (U251 or 5310) were treated with hUCBSC by intracranial injection (2.5×10 5 ). Fourteen days after hUCBSC administration, the brains were harvested, sectioned, and stained with Hematoxylin and Eosin (n≥3). Inset pictures show higher magnification at scale bar = 100 µm. (B) Characterization of hUCBSC in tumor areas in nude mice brain sections: Fourteen days after hUCBSC administration, the brains were harvested, sectioned and immunoprobed with mesenchymal stem cell markers CD29 and CD81 using Alexa flour-594 secondary antibody. (n ≥ 3). Scale bar = 100 µm. (C) Upregulation of PTEN in nude mice: Mice brain sections were immunoprobed with PTEN and CD81 using appropriate fluorescence-conjugated secondary antibodies. Secondary antibodies used for PTEN and CD81 were: goat anti-mouse Alexa flour-594 for PTEN and donkey anti-goat Alexa Fluor 488 for CD81, respectively. Scale bar = 100 µm. (D) Downregulation of XIAP in mice: Mice brain sections were probed with XIAP antibody by DAB immunohistochemistry and counterstained with DAPI. Scale bar = 100 µm. Inset pictures show DAPI. (n = > 3).

    Article Snippet: After additional PBS rinses, cells were blocked with 0.1 M PBS with 1% bovine serum albumin (BSA) for 1 h. Primary antibodies (1∶100 dilutions) specific for mesenchymal markers: mouse anti-CD29 (Millipore, Danvers, MA) and goat anti-CD81 (Santa Cruz Biotechnology, Santa Cruz, CA) and primary antibody specific for PTEN were diluted in goat serum and applied overnight at 4°C.

    Techniques: Inhibition, In Vivo, Mouse Assay, Injection, Staining, Fluorescence, Immunohistochemistry

    EGFR is required for HCVcc entry soon after CD81 binding but prior to clathrin-mediated endocytosis. (A to E) A time-of-addition experiment (depicted graphically in panel A) was performed in which Huh-7.5 cells were incubated with Jc1-Rluc HCVcc (MOI

    Journal: Journal of Virology

    Article Title: Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry

    doi: 10.1128/JVI.00750-12

    Figure Lengend Snippet: EGFR is required for HCVcc entry soon after CD81 binding but prior to clathrin-mediated endocytosis. (A to E) A time-of-addition experiment (depicted graphically in panel A) was performed in which Huh-7.5 cells were incubated with Jc1-Rluc HCVcc (MOI

    Article Snippet: For studies analyzing the effect of anti-CD81 antibody treatment on EGFR localization, the cells were incubated for 1 h at 4°C with 1:5 dilution of anti-CD81 (JS-81) or control IgG directly conjugated to FITC, washed with cold DMEM, and then shifted to 37°C for 1 h. The cells were then washed, fixed with 4% paraformaldehyde, and incubated with the anti-EGFR-Alexa 555 antibody as described above.

    Techniques: Binding Assay, Incubation

    TGF-α-mediated internalization of EGFR and CD81. (A) Huh-7.5 cells were treated with 5 nM TGF-α in the presence or absence of 5 μM erlotinib for 15 min at 37°C and fixed with paraformaldehyde, and immunofluorescence staining

    Journal: Journal of Virology

    Article Title: Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry

    doi: 10.1128/JVI.00750-12

    Figure Lengend Snippet: TGF-α-mediated internalization of EGFR and CD81. (A) Huh-7.5 cells were treated with 5 nM TGF-α in the presence or absence of 5 μM erlotinib for 15 min at 37°C and fixed with paraformaldehyde, and immunofluorescence staining

    Article Snippet: For studies analyzing the effect of anti-CD81 antibody treatment on EGFR localization, the cells were incubated for 1 h at 4°C with 1:5 dilution of anti-CD81 (JS-81) or control IgG directly conjugated to FITC, washed with cold DMEM, and then shifted to 37°C for 1 h. The cells were then washed, fixed with 4% paraformaldehyde, and incubated with the anti-EGFR-Alexa 555 antibody as described above.

    Techniques: Immunofluorescence, Staining

    Proposed model for the role of EGFR in HCV entry. HCV binding to CD81 results in cross-linking of CD81 and EGFR kinase activation, which in turn induces cointernalization of HCV-CD81-EGFR. In addition, ligand binding to EGFR activates the receptor and

    Journal: Journal of Virology

    Article Title: Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry

    doi: 10.1128/JVI.00750-12

    Figure Lengend Snippet: Proposed model for the role of EGFR in HCV entry. HCV binding to CD81 results in cross-linking of CD81 and EGFR kinase activation, which in turn induces cointernalization of HCV-CD81-EGFR. In addition, ligand binding to EGFR activates the receptor and

    Article Snippet: For studies analyzing the effect of anti-CD81 antibody treatment on EGFR localization, the cells were incubated for 1 h at 4°C with 1:5 dilution of anti-CD81 (JS-81) or control IgG directly conjugated to FITC, washed with cold DMEM, and then shifted to 37°C for 1 h. The cells were then washed, fixed with 4% paraformaldehyde, and incubated with the anti-EGFR-Alexa 555 antibody as described above.

    Techniques: Binding Assay, Activation Assay, Ligand Binding Assay

    Induction of EGFR activation by HCVcc binding to CD81. For all experiments, total EGFR was immunoprecipitated, and Western blot analyses were performed with an antibody specific for activated EGFR (phosphorylated at Tyr-1068). Total EGFR was detected

    Journal: Journal of Virology

    Article Title: Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry

    doi: 10.1128/JVI.00750-12

    Figure Lengend Snippet: Induction of EGFR activation by HCVcc binding to CD81. For all experiments, total EGFR was immunoprecipitated, and Western blot analyses were performed with an antibody specific for activated EGFR (phosphorylated at Tyr-1068). Total EGFR was detected

    Article Snippet: For studies analyzing the effect of anti-CD81 antibody treatment on EGFR localization, the cells were incubated for 1 h at 4°C with 1:5 dilution of anti-CD81 (JS-81) or control IgG directly conjugated to FITC, washed with cold DMEM, and then shifted to 37°C for 1 h. The cells were then washed, fixed with 4% paraformaldehyde, and incubated with the anti-EGFR-Alexa 555 antibody as described above.

    Techniques: Activation Assay, Binding Assay, Immunoprecipitation, Western Blot

    CD81 cross-linking induces CD81-EGFR colocalization and internalization. (A) Huh-7.5 cells were incubated with FITC-labeled control or anti-CD81 antibodies for 1 h at 4°C or shifted to 37°C for another hour. Cells were fixed and stained

    Journal: Journal of Virology

    Article Title: Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry

    doi: 10.1128/JVI.00750-12

    Figure Lengend Snippet: CD81 cross-linking induces CD81-EGFR colocalization and internalization. (A) Huh-7.5 cells were incubated with FITC-labeled control or anti-CD81 antibodies for 1 h at 4°C or shifted to 37°C for another hour. Cells were fixed and stained

    Article Snippet: For studies analyzing the effect of anti-CD81 antibody treatment on EGFR localization, the cells were incubated for 1 h at 4°C with 1:5 dilution of anti-CD81 (JS-81) or control IgG directly conjugated to FITC, washed with cold DMEM, and then shifted to 37°C for 1 h. The cells were then washed, fixed with 4% paraformaldehyde, and incubated with the anti-EGFR-Alexa 555 antibody as described above.

    Techniques: Incubation, Labeling, Staining

    Huh-7.5 cells were pretreated with erlotinib (○), lapatinib (□), anti-CD81 antibody JS-81 (◆), or anti-E2 antibody AP33 (■) and infected with either Con1/C3 HCVcc (A), Jc1 HCVcc (B), H77 HCVpp (C), Con1 HCVpp (D), or J6CF

    Journal: Journal of Virology

    Article Title: Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry

    doi: 10.1128/JVI.00750-12

    Figure Lengend Snippet: Huh-7.5 cells were pretreated with erlotinib (○), lapatinib (□), anti-CD81 antibody JS-81 (◆), or anti-E2 antibody AP33 (■) and infected with either Con1/C3 HCVcc (A), Jc1 HCVcc (B), H77 HCVpp (C), Con1 HCVpp (D), or J6CF

    Article Snippet: For studies analyzing the effect of anti-CD81 antibody treatment on EGFR localization, the cells were incubated for 1 h at 4°C with 1:5 dilution of anti-CD81 (JS-81) or control IgG directly conjugated to FITC, washed with cold DMEM, and then shifted to 37°C for 1 h. The cells were then washed, fixed with 4% paraformaldehyde, and incubated with the anti-EGFR-Alexa 555 antibody as described above.

    Techniques: Infection

    CD81 cross-linking induces EGFR activation. (A) Huh-7.5 cells were treated with increasing concentrations of anti-CD81 or anti-CLDN1 antibodies (3-fold dilutions starting at 30 μg/ml) for 1 h at 37°C. (B) EGFR activation in Huh-7.5 cells

    Journal: Journal of Virology

    Article Title: Hepatitis C Virus Induces Epidermal Growth Factor Receptor Activation via CD81 Binding for Viral Internalization and Entry

    doi: 10.1128/JVI.00750-12

    Figure Lengend Snippet: CD81 cross-linking induces EGFR activation. (A) Huh-7.5 cells were treated with increasing concentrations of anti-CD81 or anti-CLDN1 antibodies (3-fold dilutions starting at 30 μg/ml) for 1 h at 37°C. (B) EGFR activation in Huh-7.5 cells

    Article Snippet: For studies analyzing the effect of anti-CD81 antibody treatment on EGFR localization, the cells were incubated for 1 h at 4°C with 1:5 dilution of anti-CD81 (JS-81) or control IgG directly conjugated to FITC, washed with cold DMEM, and then shifted to 37°C for 1 h. The cells were then washed, fixed with 4% paraformaldehyde, and incubated with the anti-EGFR-Alexa 555 antibody as described above.

    Techniques: Activation Assay

    Conformational switch mutants modulate HCV entry. We mutated residues D196 and K201 to prevent stabilizing interactions across the EC2-TMD4 hinge. A. We performed five independent MD simulations of WT and D196A K201A CD81 in the presence of cholesterol. Images provide overlaid snapshots from representative simulations. Helix E, TMD4 and cholesterol are color coded by time. For clarity the remaining structure is shown in grey for the t=0ns snapshot only. Structures were orientated using TMD4 as a reference B. The change in angle between Helix E and TMD4, by comparison to the CD81 crystal structure, was measured over time for each D196A K201A simulation; compare to Fig 2B . C. The cumulative time spent in the open conformation for either WT or D196A K201A CD81. D. Huh-7 CD81 KO cells were transduced with lentivectors encoding WT CD81, N18A E219A (cholesterol binding mutant), D196A K201A (open mutant) or K116A D117A (closed mutant). HCV entry was assessed by challenge with a panel of HCVpp (including genotypes 1, 2, 4 and 5). HCVpp infection is shown relative to cells expressing WT CD81. Data from three representative clones and a summary plot of all HCVpp are shown. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). There was no significant difference between N18A E219A and D196A K201A. Error bars indicate standard error of the mean.

    Journal: bioRxiv

    Article Title: Cholesterol sensing by CD81 is important for hepatitis C virus entry

    doi: 10.1101/542837

    Figure Lengend Snippet: Conformational switch mutants modulate HCV entry. We mutated residues D196 and K201 to prevent stabilizing interactions across the EC2-TMD4 hinge. A. We performed five independent MD simulations of WT and D196A K201A CD81 in the presence of cholesterol. Images provide overlaid snapshots from representative simulations. Helix E, TMD4 and cholesterol are color coded by time. For clarity the remaining structure is shown in grey for the t=0ns snapshot only. Structures were orientated using TMD4 as a reference B. The change in angle between Helix E and TMD4, by comparison to the CD81 crystal structure, was measured over time for each D196A K201A simulation; compare to Fig 2B . C. The cumulative time spent in the open conformation for either WT or D196A K201A CD81. D. Huh-7 CD81 KO cells were transduced with lentivectors encoding WT CD81, N18A E219A (cholesterol binding mutant), D196A K201A (open mutant) or K116A D117A (closed mutant). HCV entry was assessed by challenge with a panel of HCVpp (including genotypes 1, 2, 4 and 5). HCVpp infection is shown relative to cells expressing WT CD81. Data from three representative clones and a summary plot of all HCVpp are shown. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). There was no significant difference between N18A E219A and D196A K201A. Error bars indicate standard error of the mean.

    Article Snippet: Nuclear debris was pelleted at 12000g for 10 minutes at 4°C followed by co-immunoprecipitation using Pierce Crosslink Immunoprecipitation kit (Thermo Fisher Scientific, Waltham, MA, USA) with crosslinked anti-CD81 (clone 1.3.3.22, Santa Cruz).

    Techniques: Transduction, Binding Assay, Mutagenesis, Infection, Expressing, Clone Assay

    Cell surface functionality of CD81 mutants. Huh-7 CD81 KO cells were co-transduced with lentivectors encoding human CD19 and CD81 or empty vector. A. Representative flow cytometry histograms, all samples received CD19 lentivector plus the indicated CD81/control vector. The plot on the left demonstrates CD81 surface expression (i), the right-hand plot displays CD81-dependent trafficking of CD19 to the cell surface (ii). B. CD81 expression on CHO cells confers binding on soluble HCV E2. The plot on the left demonstrates CD81 surface expression (i), the right-hand plot displays sE2 binding to transduced CHO cells (ii). C. Quantification of sE2 binding expressed relative to WT CD81. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). Error bars indicate standard error of the mean.

    Journal: bioRxiv

    Article Title: Cholesterol sensing by CD81 is important for hepatitis C virus entry

    doi: 10.1101/542837

    Figure Lengend Snippet: Cell surface functionality of CD81 mutants. Huh-7 CD81 KO cells were co-transduced with lentivectors encoding human CD19 and CD81 or empty vector. A. Representative flow cytometry histograms, all samples received CD19 lentivector plus the indicated CD81/control vector. The plot on the left demonstrates CD81 surface expression (i), the right-hand plot displays CD81-dependent trafficking of CD19 to the cell surface (ii). B. CD81 expression on CHO cells confers binding on soluble HCV E2. The plot on the left demonstrates CD81 surface expression (i), the right-hand plot displays sE2 binding to transduced CHO cells (ii). C. Quantification of sE2 binding expressed relative to WT CD81. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). Error bars indicate standard error of the mean.

    Article Snippet: Nuclear debris was pelleted at 12000g for 10 minutes at 4°C followed by co-immunoprecipitation using Pierce Crosslink Immunoprecipitation kit (Thermo Fisher Scientific, Waltham, MA, USA) with crosslinked anti-CD81 (clone 1.3.3.22, Santa Cruz).

    Techniques: Transduction, Plasmid Preparation, Flow Cytometry, Expressing, Binding Assay

    Cholesterol sensing is important for authentic HCV infection. Huh-7 CD81 KO cells were transduced with lentivectors expressing the stated CD81 mutants and were then challenged with J6/JFH HCVcc. A. Representative micrographs of HCVcc infection in transduced cells; DAPI nuclei shown in blue, viral antigen NS5A displayed in orange, scale bar = 100 μ m. B. Quantification of infection, compiled from four independent experiments, data is expressed relative to infection in cells expressing WT CD81. C. Huh-7 Lunet N cells stably expressing the stated CD81 mutants were challenged with a panel of diverse HCVcc bearing the glycoproteins of genotypes 1, 2, 3, 4 and 5. Infection was quantified via a virally encoded luciferase reporter and is expressed relative to WT CD81. Data from three representative clones and a summary plot of all HCVcc are shown. Asterisks indicate statistical significance from WT (n=3, one-way ANOVA, Prism). Error bars indicate standard error of the mean.

    Journal: bioRxiv

    Article Title: Cholesterol sensing by CD81 is important for hepatitis C virus entry

    doi: 10.1101/542837

    Figure Lengend Snippet: Cholesterol sensing is important for authentic HCV infection. Huh-7 CD81 KO cells were transduced with lentivectors expressing the stated CD81 mutants and were then challenged with J6/JFH HCVcc. A. Representative micrographs of HCVcc infection in transduced cells; DAPI nuclei shown in blue, viral antigen NS5A displayed in orange, scale bar = 100 μ m. B. Quantification of infection, compiled from four independent experiments, data is expressed relative to infection in cells expressing WT CD81. C. Huh-7 Lunet N cells stably expressing the stated CD81 mutants were challenged with a panel of diverse HCVcc bearing the glycoproteins of genotypes 1, 2, 3, 4 and 5. Infection was quantified via a virally encoded luciferase reporter and is expressed relative to WT CD81. Data from three representative clones and a summary plot of all HCVcc are shown. Asterisks indicate statistical significance from WT (n=3, one-way ANOVA, Prism). Error bars indicate standard error of the mean.

    Article Snippet: Nuclear debris was pelleted at 12000g for 10 minutes at 4°C followed by co-immunoprecipitation using Pierce Crosslink Immunoprecipitation kit (Thermo Fisher Scientific, Waltham, MA, USA) with crosslinked anti-CD81 (clone 1.3.3.22, Santa Cruz).

    Techniques: Infection, Transduction, Expressing, Stable Transfection, Luciferase, Clone Assay

    Conformational switch mutants exhibit altered protein interaction networks. A. Volcano plot visualizing differences from co-IPs of Huh-7 Lunet N CD81 WT versus Lunet N control cells (n=4 biological replicates for each cell line). LFQ intensity differences (log2) are plotted against the t-test p value (−logP). Significant interactors were defined by a permutation-based FDR using S0=1 as described [ 94 ]. Reference proteins (CD81, SCARB1, CLDN1, EGFR, TFRC, CAPN5 ITGB and CD151) are highlighted, color coded as in B. B. Mean LFQ intensity differences (log2) of interactors in CD81 co-IP (Huh-7 Lunet N CD81 WT and mutants versus Lunet N control cells). Error bars indicate standard error of the mean (n=4) C. Venn diagrams showing the overlap of significantly enriched proteins found in CD81 co-IPs from WT in grey, N18A E219A (Chl) in orange, D196A K201A (O) in purple and K116A D117A (C) in green. Values below each title indicate significant interactors for each CD81 variant, values in the center of each Venn diagram indicate overlapping interactors.

    Journal: bioRxiv

    Article Title: Cholesterol sensing by CD81 is important for hepatitis C virus entry

    doi: 10.1101/542837

    Figure Lengend Snippet: Conformational switch mutants exhibit altered protein interaction networks. A. Volcano plot visualizing differences from co-IPs of Huh-7 Lunet N CD81 WT versus Lunet N control cells (n=4 biological replicates for each cell line). LFQ intensity differences (log2) are plotted against the t-test p value (−logP). Significant interactors were defined by a permutation-based FDR using S0=1 as described [ 94 ]. Reference proteins (CD81, SCARB1, CLDN1, EGFR, TFRC, CAPN5 ITGB and CD151) are highlighted, color coded as in B. B. Mean LFQ intensity differences (log2) of interactors in CD81 co-IP (Huh-7 Lunet N CD81 WT and mutants versus Lunet N control cells). Error bars indicate standard error of the mean (n=4) C. Venn diagrams showing the overlap of significantly enriched proteins found in CD81 co-IPs from WT in grey, N18A E219A (Chl) in orange, D196A K201A (O) in purple and K116A D117A (C) in green. Values below each title indicate significant interactors for each CD81 variant, values in the center of each Venn diagram indicate overlapping interactors.

    Article Snippet: Nuclear debris was pelleted at 12000g for 10 minutes at 4°C followed by co-immunoprecipitation using Pierce Crosslink Immunoprecipitation kit (Thermo Fisher Scientific, Waltham, MA, USA) with crosslinked anti-CD81 (clone 1.3.3.22, Santa Cruz).

    Techniques: Co-Immunoprecipitation Assay, Variant Assay

    Conformational switching of CD81 in the absence of cholesterol. We performed five independent 500ns MD simulations of WT CD81 with and without cholesterol. A. Snapshots summarising representative simulations from either condition. The Δ° measurement reflects the change in the angle between helix E of the EC2 and TMD4 (as annotated), by comparison to the CD81 crystal structure. For each snapshot the region from which the measurement was taken is color-coded by time. Cholesterol is shown in red. Structures were orientated using TMD4 as a reference. Examples of the orientation of D196 and K201 are shown as insets. B. The angle between helix E and TMD4 was measured over time for each simulation, 25° was chosen as a threshold to indicate conformational switching. C. The cumulative time spent in the open conformation was calculated across all simulations for either experimental condition. D. The distance between D196 K201 was measured over time for each simulation, the dashed line indicates the distance under which electrostatic interactions and hydrogen bonding occurs (10Å). E. The average distance between D196 and K201 with and without cholesterol, data points represent the mean value for each simulation, asterisk indicates statistical significance (n=5 simulations, unpaired T-test, Prism).

    Journal: bioRxiv

    Article Title: Cholesterol sensing by CD81 is important for hepatitis C virus entry

    doi: 10.1101/542837

    Figure Lengend Snippet: Conformational switching of CD81 in the absence of cholesterol. We performed five independent 500ns MD simulations of WT CD81 with and without cholesterol. A. Snapshots summarising representative simulations from either condition. The Δ° measurement reflects the change in the angle between helix E of the EC2 and TMD4 (as annotated), by comparison to the CD81 crystal structure. For each snapshot the region from which the measurement was taken is color-coded by time. Cholesterol is shown in red. Structures were orientated using TMD4 as a reference. Examples of the orientation of D196 and K201 are shown as insets. B. The angle between helix E and TMD4 was measured over time for each simulation, 25° was chosen as a threshold to indicate conformational switching. C. The cumulative time spent in the open conformation was calculated across all simulations for either experimental condition. D. The distance between D196 K201 was measured over time for each simulation, the dashed line indicates the distance under which electrostatic interactions and hydrogen bonding occurs (10Å). E. The average distance between D196 and K201 with and without cholesterol, data points represent the mean value for each simulation, asterisk indicates statistical significance (n=5 simulations, unpaired T-test, Prism).

    Article Snippet: Nuclear debris was pelleted at 12000g for 10 minutes at 4°C followed by co-immunoprecipitation using Pierce Crosslink Immunoprecipitation kit (Thermo Fisher Scientific, Waltham, MA, USA) with crosslinked anti-CD81 (clone 1.3.3.22, Santa Cruz).

    Techniques:

    Mutations in the cholesterol binding pocket of CD81 modulate HCV entry. A. Cholesterol (red) is coordinated in the intramembrane cavity of CD81 by hydrogen bonds with inward facing residues N18 and E219. We made various mutations at these sites to disrupt this interaction. B. The cholesterol molecule sits in the centre of an intramembrane binding pocket. In the V68W M72W A108W V212W mutant this space is occupied by tryptophan residues (blue residues). C. The cell surface expression levels of each mutant CD81 was assessed by flow cytometry. D. Huh-7 CD81 KO cells were transduced with lentivector encoding WT CD81 or empty vector control. The cells were surface labelled with anti-CD81 mAb and lysed in Brij-98 detergent buffer. CD81-mAb complexes were pulled-down with protein G beads and associated free cholesterol was measured. Our positive control demonstrates the accuracy of the assay. The dashed line indicates the limit of detection E. We assessed cholesterol association with WT and mutant CD81. Data is expressed relative to WT CD81, asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). A representative western blot demonstrating equivalent levels of CD81 in the whole cell lysate (WCL) and pull-down (IP) F. Huh-7 CD81 KO cells were transduced with lentivectors encoding WT and mutant CD81. HCV entry was assessed by challenge with a panel of HCVpp (including genotypes 1, 2 and 5). HCVpp infection is shown relative to cells expressing WT CD81. Data from three representative clones and a summary plot of all HCVpp are shown. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). Error bars indicate standard error of the mean.

    Journal: bioRxiv

    Article Title: Cholesterol sensing by CD81 is important for hepatitis C virus entry

    doi: 10.1101/542837

    Figure Lengend Snippet: Mutations in the cholesterol binding pocket of CD81 modulate HCV entry. A. Cholesterol (red) is coordinated in the intramembrane cavity of CD81 by hydrogen bonds with inward facing residues N18 and E219. We made various mutations at these sites to disrupt this interaction. B. The cholesterol molecule sits in the centre of an intramembrane binding pocket. In the V68W M72W A108W V212W mutant this space is occupied by tryptophan residues (blue residues). C. The cell surface expression levels of each mutant CD81 was assessed by flow cytometry. D. Huh-7 CD81 KO cells were transduced with lentivector encoding WT CD81 or empty vector control. The cells were surface labelled with anti-CD81 mAb and lysed in Brij-98 detergent buffer. CD81-mAb complexes were pulled-down with protein G beads and associated free cholesterol was measured. Our positive control demonstrates the accuracy of the assay. The dashed line indicates the limit of detection E. We assessed cholesterol association with WT and mutant CD81. Data is expressed relative to WT CD81, asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). A representative western blot demonstrating equivalent levels of CD81 in the whole cell lysate (WCL) and pull-down (IP) F. Huh-7 CD81 KO cells were transduced with lentivectors encoding WT and mutant CD81. HCV entry was assessed by challenge with a panel of HCVpp (including genotypes 1, 2 and 5). HCVpp infection is shown relative to cells expressing WT CD81. Data from three representative clones and a summary plot of all HCVpp are shown. Asterisks indicate statistical significance from WT (n=4, one-way ANOVA, Prism). Error bars indicate standard error of the mean.

    Article Snippet: Nuclear debris was pelleted at 12000g for 10 minutes at 4°C followed by co-immunoprecipitation using Pierce Crosslink Immunoprecipitation kit (Thermo Fisher Scientific, Waltham, MA, USA) with crosslinked anti-CD81 (clone 1.3.3.22, Santa Cruz).

    Techniques: Binding Assay, Mutagenesis, Expressing, Flow Cytometry, Transduction, Plasmid Preparation, Positive Control, Western Blot, Infection, Clone Assay