Structured Review

Millipore cd81
Characterization of exosomes isolated from A549 cells (Exo-A549) or ZIKV infected A549 cells (Exo-ZIKV). Transmission electron micrograph of Exo-A549 (A) and Exo-ZIKV (B). Nanoparticle tracking analysis of exosomes indicated Exo-A549 particles sizes median diameter of 125.3 nm (C) and Exo-ZIKV median size diameter of 127.2 nm (D). Peak analysis of exosomes (E). Western blotting of exosomes was performed to confirm the presence of exosomal marker protein, CD63, <t>CD81</t> and TSG101. Absence the endoplasmic reticulum protein, Calnexin, in exosomes but was detectable in whole cell lysates (F).
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Images

1) Product Images from "DEFA1B inhibits ZIKV replication and retards cell cycle progression through interaction with ORC1"

Article Title: DEFA1B inhibits ZIKV replication and retards cell cycle progression through interaction with ORC1

Journal: Life Sciences

doi: 10.1016/j.lfs.2020.118564

Characterization of exosomes isolated from A549 cells (Exo-A549) or ZIKV infected A549 cells (Exo-ZIKV). Transmission electron micrograph of Exo-A549 (A) and Exo-ZIKV (B). Nanoparticle tracking analysis of exosomes indicated Exo-A549 particles sizes median diameter of 125.3 nm (C) and Exo-ZIKV median size diameter of 127.2 nm (D). Peak analysis of exosomes (E). Western blotting of exosomes was performed to confirm the presence of exosomal marker protein, CD63, CD81 and TSG101. Absence the endoplasmic reticulum protein, Calnexin, in exosomes but was detectable in whole cell lysates (F).
Figure Legend Snippet: Characterization of exosomes isolated from A549 cells (Exo-A549) or ZIKV infected A549 cells (Exo-ZIKV). Transmission electron micrograph of Exo-A549 (A) and Exo-ZIKV (B). Nanoparticle tracking analysis of exosomes indicated Exo-A549 particles sizes median diameter of 125.3 nm (C) and Exo-ZIKV median size diameter of 127.2 nm (D). Peak analysis of exosomes (E). Western blotting of exosomes was performed to confirm the presence of exosomal marker protein, CD63, CD81 and TSG101. Absence the endoplasmic reticulum protein, Calnexin, in exosomes but was detectable in whole cell lysates (F).

Techniques Used: Isolation, Infection, Transmission Assay, Western Blot, Marker

2) Product Images from "Improvement of sensory neuron growth and survival via negatively regulating PTEN by miR-21-5p-contained small extracellular vesicles from skin precursor-derived Schwann cells"

Article Title: Improvement of sensory neuron growth and survival via negatively regulating PTEN by miR-21-5p-contained small extracellular vesicles from skin precursor-derived Schwann cells

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-020-02125-4

Characterization of SKP-SC-derived EVs. a Representative TEM image of SKP-SC-EVs presenting a typical cup-like concavity shape. Scale bar, 200 nm. b Representative traces from nanoparticle tracking analysis for SKP-SC-EVs. c Western blots showing the positive expression of exosomal markers CD9, CD63, CD81, and TSG101 in EVs, and β-actin and S100β were used as control markers in SKP-SCs. d PKH67 labeled EVs (green) were showing within the cytoplasm and axon of TUJ1 positive sensory neurons (red), and nuclei were labeled with DAPI (blue). Scale bar, 30 μm
Figure Legend Snippet: Characterization of SKP-SC-derived EVs. a Representative TEM image of SKP-SC-EVs presenting a typical cup-like concavity shape. Scale bar, 200 nm. b Representative traces from nanoparticle tracking analysis for SKP-SC-EVs. c Western blots showing the positive expression of exosomal markers CD9, CD63, CD81, and TSG101 in EVs, and β-actin and S100β were used as control markers in SKP-SCs. d PKH67 labeled EVs (green) were showing within the cytoplasm and axon of TUJ1 positive sensory neurons (red), and nuclei were labeled with DAPI (blue). Scale bar, 30 μm

Techniques Used: Derivative Assay, Transmission Electron Microscopy, Western Blot, Expressing, Labeling

3) Product Images from "CD81 association with SAMHD1 enhances HIV-1 reverse transcription by increasing dNTP levels"

Article Title: CD81 association with SAMHD1 enhances HIV-1 reverse transcription by increasing dNTP levels

Journal: Nature microbiology

doi: 10.1038/s41564-017-0019-0

CD81 regulates SAMHD1 expression. a) Primary T lymphoblasts transfected with control or CD81 siRNA, and Hela/R5 cells transfected or not with CRISPR/Cas9-CD81 were lysed and immunoblotted for CD81, SAMHD1 and phosphorylated SAMHD1. Tubulin or cofilin were used as loading controls. Blots are from representative experiments out of 2 (lymphoblasts) and 3 (Hela/R5). Signal ratios in relation to the loading controls are depicted. Arrows indicate the SAMHD1 band that has the predicted molecular weight (~70kDa). b) Hela/R5 cells transfected or not with CRISPR/Cas9-CD81 were fixed, permeabilized, immunolabelled for SAMHD1 and CD81, and analysed by flow cytometry. Histograms show a representative experiment out of 3. The negative control corresponds to cells stained only with secondary antibody. c-d) CRISPR/Cas9-CD81 or control Hela/R5 cells were treated for 6h with the vehicle or with the depicted concentrations of (c) MG132 or (d) NH 4 Cl. Cells were lysed and immunoblotted for SAMHD1, and ERM as loading control. Graph shows the mean fold change ± SEM of SAMHD1/ERM signal ratio from (c) 3 or (d) 2 independent experiments analysed by one-way ANOVA with Bonferroni’s post-test.
Figure Legend Snippet: CD81 regulates SAMHD1 expression. a) Primary T lymphoblasts transfected with control or CD81 siRNA, and Hela/R5 cells transfected or not with CRISPR/Cas9-CD81 were lysed and immunoblotted for CD81, SAMHD1 and phosphorylated SAMHD1. Tubulin or cofilin were used as loading controls. Blots are from representative experiments out of 2 (lymphoblasts) and 3 (Hela/R5). Signal ratios in relation to the loading controls are depicted. Arrows indicate the SAMHD1 band that has the predicted molecular weight (~70kDa). b) Hela/R5 cells transfected or not with CRISPR/Cas9-CD81 were fixed, permeabilized, immunolabelled for SAMHD1 and CD81, and analysed by flow cytometry. Histograms show a representative experiment out of 3. The negative control corresponds to cells stained only with secondary antibody. c-d) CRISPR/Cas9-CD81 or control Hela/R5 cells were treated for 6h with the vehicle or with the depicted concentrations of (c) MG132 or (d) NH 4 Cl. Cells were lysed and immunoblotted for SAMHD1, and ERM as loading control. Graph shows the mean fold change ± SEM of SAMHD1/ERM signal ratio from (c) 3 or (d) 2 independent experiments analysed by one-way ANOVA with Bonferroni’s post-test.

Techniques Used: Expressing, Transfection, CRISPR, Molecular Weight, Flow Cytometry, Cytometry, Negative Control, Staining

CD81 expression supports R5-tropic HIV-1 RT. a) Time course of HIV-1 RT measured by qPCR of early or late RT products at 24 or 48h post-infection. Hela/R5 expressing GFP, CD81GFP or CD81∆cytGFP were infected with HIV-1 (BaL) or HIV-VSV-G. Data are mean fold change ± SEM from 4 independent experiments performed in triplicate, and analysed by two-way ANOVA with Bonferroni’s post-test. b) Hela/R5 cells expressing GFP, CD81GFP or CD81∆cytGFP were infected with single-cycle HIV-1-R5-Luc or HIV-VSV-G-Luc. Data are mean fold-induction ± SEM of luciferase activity from 3 independent experiments performed in triplicate, and analysed by one-way ANOVA with Tukey’s post-test. c) Hela/R5 cells transfected with CRISPR/Cas9-CD81 or left untreated were infected and analysed as in b . Data are mean fold change ± SEM from 3 independent experiments performed in triplicate, and analysed by paired Student t -test, *** p
Figure Legend Snippet: CD81 expression supports R5-tropic HIV-1 RT. a) Time course of HIV-1 RT measured by qPCR of early or late RT products at 24 or 48h post-infection. Hela/R5 expressing GFP, CD81GFP or CD81∆cytGFP were infected with HIV-1 (BaL) or HIV-VSV-G. Data are mean fold change ± SEM from 4 independent experiments performed in triplicate, and analysed by two-way ANOVA with Bonferroni’s post-test. b) Hela/R5 cells expressing GFP, CD81GFP or CD81∆cytGFP were infected with single-cycle HIV-1-R5-Luc or HIV-VSV-G-Luc. Data are mean fold-induction ± SEM of luciferase activity from 3 independent experiments performed in triplicate, and analysed by one-way ANOVA with Tukey’s post-test. c) Hela/R5 cells transfected with CRISPR/Cas9-CD81 or left untreated were infected and analysed as in b . Data are mean fold change ± SEM from 3 independent experiments performed in triplicate, and analysed by paired Student t -test, *** p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Infection, Luciferase, Activity Assay, Transfection, CRISPR

CD81 regulates X4-tropic HIV-1 RT. a) Primary T lymphoblasts pre-treated with 2μM of scramble or CD81pept for 5 days, were infected with HIV-1 NL4-3 strain or HIV-VSV-G. Early or late RT products were measured as in Figure 2a . Data are mean fold change ± SEM of 2 independent experiments performed in triplicate, and analysed by two-way ANOVA with Bonferroni’s post-test. b) Primary T lymphoblasts transfected with control or CD81 siRNA were infected with NL4-3 strain, and RT was measured as in Figure 2a . Data are from a representative experiment out of two. In-box shows immunoblots of whole cell lysates from the represented experiment probed for CD81, and ERM as loading control. The CD81/ERM signal ratio is indicated.
Figure Legend Snippet: CD81 regulates X4-tropic HIV-1 RT. a) Primary T lymphoblasts pre-treated with 2μM of scramble or CD81pept for 5 days, were infected with HIV-1 NL4-3 strain or HIV-VSV-G. Early or late RT products were measured as in Figure 2a . Data are mean fold change ± SEM of 2 independent experiments performed in triplicate, and analysed by two-way ANOVA with Bonferroni’s post-test. b) Primary T lymphoblasts transfected with control or CD81 siRNA were infected with NL4-3 strain, and RT was measured as in Figure 2a . Data are from a representative experiment out of two. In-box shows immunoblots of whole cell lysates from the represented experiment probed for CD81, and ERM as loading control. The CD81/ERM signal ratio is indicated.

Techniques Used: Infection, Transfection, Western Blot

CD81 negatively regulates cellular dNTP content through SAMHD1. a) Jurkat T cells transfected with control or CD81 siRNA were infected with HIV-1 NL4-3 strain, and early RT products were measured by qPCR at the indicated times. Data are mean fold change ± SEM of 2 independent experiments performed in triplicate. In-box shows representative immunoblots of CD81 and tubulin as loading control, and the CD81/Tubulin signal ratio is indicated. b) Jurkat T cells pre-treated with 2μM of scramble or CD81pept for 5 days were infected with NL4-3 strain, and RT was analysed as in a . c) Mean fold change ± SEM of the dNTP content of Hela/R5 or Hela/R5 CRISPR/Cas9-CD81 cells (left graph, 4 independent experiments), and primary T lymphoblasts transfected with control or CD81 siRNA (right graph, 2 independent experiments) measured by a HIV RT-based dNTP assay. Analysis was performed by paired Student t -test, *** p
Figure Legend Snippet: CD81 negatively regulates cellular dNTP content through SAMHD1. a) Jurkat T cells transfected with control or CD81 siRNA were infected with HIV-1 NL4-3 strain, and early RT products were measured by qPCR at the indicated times. Data are mean fold change ± SEM of 2 independent experiments performed in triplicate. In-box shows representative immunoblots of CD81 and tubulin as loading control, and the CD81/Tubulin signal ratio is indicated. b) Jurkat T cells pre-treated with 2μM of scramble or CD81pept for 5 days were infected with NL4-3 strain, and RT was analysed as in a . c) Mean fold change ± SEM of the dNTP content of Hela/R5 or Hela/R5 CRISPR/Cas9-CD81 cells (left graph, 4 independent experiments), and primary T lymphoblasts transfected with control or CD81 siRNA (right graph, 2 independent experiments) measured by a HIV RT-based dNTP assay. Analysis was performed by paired Student t -test, *** p

Techniques Used: Transfection, Infection, Real-time Polymerase Chain Reaction, Western Blot, CRISPR

SAMHD1 is partially enriched at early endosomes. a) Hela/R5 cells were transfected with control or CD81 siRNA, adhered for 4h onto fibronectin (FN), fixed, permeabilized in PBS 0.1% Triton X-100 for 5min, and immunolabelled for SAMHD1. Images show one single confocal plane, nuclei are in blue. Arrows indicate SAMHD1 accumulation in circular-shaped intracellular structures, bars = 10μm. Graphs show means ± SEM of the number (counts/cell) and area (μm 2 /cell) of the cytoplasmic structures observed (n=230 cells, 4 independent experiments analysed by Student t -test, *** p = 0.0005 (upper) and *** p = 0.0006 (bottom)). b) Hela/R5 cells treated with 2μM of scramble or CD81pept were analysed as in a (n=400 cells, 4 independent experiments analysed by Student t -test, ** p = 0.0063 (upper) and ** p = 0.0054 (bottom)). c) Hela/R5 cells transfected with GFP, CD81GFP or CD81∆cytGFP were analysed as in a (n=20 cells, 2 independent experiments analysed by one-way ANOVA with Tukey’s post-test). d) Hela/R5 transfected with control or CD81 siRNA were treated as in a . Images show SAMHD1 (green), EEA1 (red), LAMP-1 (magenta), nuclei (blue), DIC, SAMHD1/EEA1 co-localization channel (white), and merged images. One single confocal plane is shown, bars = 10μm. Graphs represent the quantification of SAMHD1-EEA1 co-localization performed in 3D stack confocal microscopy images, showing means ± SEM of the Pearson’s coefficient; and of the % of SAMHD1 signal co-localized with EEA1 signal with respect to the total SAMHD1 signal in the cell (n = 200 cells, 3 independent experiments analysed by Student t -test, * p = 0.0262 (left) and * p = 0.0479 (right)).
Figure Legend Snippet: SAMHD1 is partially enriched at early endosomes. a) Hela/R5 cells were transfected with control or CD81 siRNA, adhered for 4h onto fibronectin (FN), fixed, permeabilized in PBS 0.1% Triton X-100 for 5min, and immunolabelled for SAMHD1. Images show one single confocal plane, nuclei are in blue. Arrows indicate SAMHD1 accumulation in circular-shaped intracellular structures, bars = 10μm. Graphs show means ± SEM of the number (counts/cell) and area (μm 2 /cell) of the cytoplasmic structures observed (n=230 cells, 4 independent experiments analysed by Student t -test, *** p = 0.0005 (upper) and *** p = 0.0006 (bottom)). b) Hela/R5 cells treated with 2μM of scramble or CD81pept were analysed as in a (n=400 cells, 4 independent experiments analysed by Student t -test, ** p = 0.0063 (upper) and ** p = 0.0054 (bottom)). c) Hela/R5 cells transfected with GFP, CD81GFP or CD81∆cytGFP were analysed as in a (n=20 cells, 2 independent experiments analysed by one-way ANOVA with Tukey’s post-test). d) Hela/R5 transfected with control or CD81 siRNA were treated as in a . Images show SAMHD1 (green), EEA1 (red), LAMP-1 (magenta), nuclei (blue), DIC, SAMHD1/EEA1 co-localization channel (white), and merged images. One single confocal plane is shown, bars = 10μm. Graphs represent the quantification of SAMHD1-EEA1 co-localization performed in 3D stack confocal microscopy images, showing means ± SEM of the Pearson’s coefficient; and of the % of SAMHD1 signal co-localized with EEA1 signal with respect to the total SAMHD1 signal in the cell (n = 200 cells, 3 independent experiments analysed by Student t -test, * p = 0.0262 (left) and * p = 0.0479 (right)).

Techniques Used: Transfection, Confocal Microscopy

The C-terminal domain of CD81 mediates its association with SAMHD1. a) SAMHD1 immunoblot of primary T lymphoblast lysates pulled-down with biotinylated peptides of tetraspanins CD81, CD9 and CD151 C-terminal domains. Sepharose-negative control and whole cell lysate are shown. b) Primary T lymphoblast lysates were immunoprecipitated and immunoblotted with SAMHD1 or CD81 antibodies. Control beads incubated with cell lysate, and whole cell lysate are shown. c) Hela/R5 cells or primary T lymphoblasts were plated onto PLL, fixed, permeabilized in PBS 0.5% Triton X-100 for 5 min, stained for CD81 (red) and SAMHD1 (green), and analysed by confocal microscopy. One single confocal plane is shown, nuclei are in blue. Bar = 10μm. d) Hela/R5 cells transfected with control siRNA (siControl) or CD81 siRNA (siCD81) were plated onto PLL (10μg/ml), anti-CD9 (VJ1/20, 10μg/ml), anti-CD4 (HP2/6, 10μg/ml) or anti-CD81 (5A6, 10μg/ml) monoclonal antibodies for 2h, fixed, permeabilized in 0.5% Triton X-100 for 5min, and stained for SAMHD1 (polyclonal antibody). Images show a single confocal plane at a ventral position, bar = 10μm. Graph shows means ± SEM of the number (counts/cell) of SAMHD1 + clusters (n=50 cells, 3 independent experiments), analysed by one-way ANOVA with Tukey’s post-test. e) Duo-link immunoassay of primary T lymphoblasts plated onto PLL, permeabilized in PBS 0.5% Triton X-100 for 5min, and stained for SAMHD1 and CD81. SAMHD1/CD147 and CD81/ERM were used as negative and positive controls respectively, bar = 10μm. Graph shows the number of dots per cell; each dot represents a single cell, bars denote the mean of scatter plots, and data was analysed by one-way ANOVA with Dunns post-test.
Figure Legend Snippet: The C-terminal domain of CD81 mediates its association with SAMHD1. a) SAMHD1 immunoblot of primary T lymphoblast lysates pulled-down with biotinylated peptides of tetraspanins CD81, CD9 and CD151 C-terminal domains. Sepharose-negative control and whole cell lysate are shown. b) Primary T lymphoblast lysates were immunoprecipitated and immunoblotted with SAMHD1 or CD81 antibodies. Control beads incubated with cell lysate, and whole cell lysate are shown. c) Hela/R5 cells or primary T lymphoblasts were plated onto PLL, fixed, permeabilized in PBS 0.5% Triton X-100 for 5 min, stained for CD81 (red) and SAMHD1 (green), and analysed by confocal microscopy. One single confocal plane is shown, nuclei are in blue. Bar = 10μm. d) Hela/R5 cells transfected with control siRNA (siControl) or CD81 siRNA (siCD81) were plated onto PLL (10μg/ml), anti-CD9 (VJ1/20, 10μg/ml), anti-CD4 (HP2/6, 10μg/ml) or anti-CD81 (5A6, 10μg/ml) monoclonal antibodies for 2h, fixed, permeabilized in 0.5% Triton X-100 for 5min, and stained for SAMHD1 (polyclonal antibody). Images show a single confocal plane at a ventral position, bar = 10μm. Graph shows means ± SEM of the number (counts/cell) of SAMHD1 + clusters (n=50 cells, 3 independent experiments), analysed by one-way ANOVA with Tukey’s post-test. e) Duo-link immunoassay of primary T lymphoblasts plated onto PLL, permeabilized in PBS 0.5% Triton X-100 for 5min, and stained for SAMHD1 and CD81. SAMHD1/CD147 and CD81/ERM were used as negative and positive controls respectively, bar = 10μm. Graph shows the number of dots per cell; each dot represents a single cell, bars denote the mean of scatter plots, and data was analysed by one-way ANOVA with Dunns post-test.

Techniques Used: Negative Control, Immunoprecipitation, Incubation, Staining, Confocal Microscopy, Transfection

4) Product Images from "Extracellular vesicles from skin precursor-derived Schwann cells promote axonal outgrowth and regeneration of motoneurons via Akt/mTOR/p70S6K pathway"

Article Title: Extracellular vesicles from skin precursor-derived Schwann cells promote axonal outgrowth and regeneration of motoneurons via Akt/mTOR/p70S6K pathway

Journal: Annals of Translational Medicine

doi: 10.21037/atm-20-5965

Characterization of EVs derived from SKP-SCs. (A) Representative size distribution of EVs derived from SKP-SCs detected by nanoparticle tracking analysis (NTA). (B) The typical cup-shape of EVs was showed under transmission electron microscopy (TEM). Scale bar, 200 nm. (C) Representative western blot analysis showed the positive expression of CD9, CD63, CD81, HSP70, and TSG101 in SKP-SC-EVs, SKP-SC was used as control. (D) Motoneurons were incubated with PKH67-labeled EVs or PBS (as control), photomicrographs showed the intake of PKH67-labeled EV particles (green) by TUJ1 stained motoneurons (red) with DAPI-stained nuclei (blue). Scale bar, 25 µm. The magnification image showed that the internalized EVs distributed in both soma of cytoplasm and axons of motoneurons. Scale bar, 75 µm. SKP, skin precursor; SC, Schwann cell; EV, extracellular vesicle; PBS, phosphate buffered saline; TUJ1, β-tubulin3; DAPI, 4',6-diamidino-2-phenylindole.
Figure Legend Snippet: Characterization of EVs derived from SKP-SCs. (A) Representative size distribution of EVs derived from SKP-SCs detected by nanoparticle tracking analysis (NTA). (B) The typical cup-shape of EVs was showed under transmission electron microscopy (TEM). Scale bar, 200 nm. (C) Representative western blot analysis showed the positive expression of CD9, CD63, CD81, HSP70, and TSG101 in SKP-SC-EVs, SKP-SC was used as control. (D) Motoneurons were incubated with PKH67-labeled EVs or PBS (as control), photomicrographs showed the intake of PKH67-labeled EV particles (green) by TUJ1 stained motoneurons (red) with DAPI-stained nuclei (blue). Scale bar, 25 µm. The magnification image showed that the internalized EVs distributed in both soma of cytoplasm and axons of motoneurons. Scale bar, 75 µm. SKP, skin precursor; SC, Schwann cell; EV, extracellular vesicle; PBS, phosphate buffered saline; TUJ1, β-tubulin3; DAPI, 4',6-diamidino-2-phenylindole.

Techniques Used: Derivative Assay, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Western Blot, Expressing, Incubation, Labeling, Staining

5) Product Images from "Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *"

Article Title: Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.606269

EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized
Figure Legend Snippet: EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized

Techniques Used: Staining

CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells
Figure Legend Snippet: CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells

Techniques Used: Inhibition

6) Product Images from "Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *"

Article Title: Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.606269

EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized
Figure Legend Snippet: EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized

Techniques Used: Staining

CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells
Figure Legend Snippet: CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells

Techniques Used: Inhibition

7) Product Images from "Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *"

Article Title: Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.606269

EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized
Figure Legend Snippet: EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized

Techniques Used: Staining

CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells
Figure Legend Snippet: CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells

Techniques Used: Inhibition

8) Product Images from "Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *"

Article Title: Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.606269

EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized
Figure Legend Snippet: EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized

Techniques Used: Staining

CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells
Figure Legend Snippet: CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells

Techniques Used: Inhibition

9) Product Images from "Pseudotyping retrovirus like particles vaccine candidates with Hepatitis C virus envelope protein E2 requires the cellular expression of CD81"

Article Title: Pseudotyping retrovirus like particles vaccine candidates with Hepatitis C virus envelope protein E2 requires the cellular expression of CD81

Journal: AMB Express

doi: 10.1186/s13568-019-0741-5

Cellular expression of and incorporation of HCV envelope proteins in chimeric HCVpp. a Cellular expression of MLV p30, CD81, HCV E1 and HCV E2 determined by western blotting in 293rVLP, 293rVLP-HCVpp, 293rVLP-HCVpp (AEE), 293rVLP-shCD81 cells and 293rVLP-HCVpp-shCD81 (BEE); b incorporation of CD81, HCV E1 and HCV E2 membrane proteins in null retrovirus particles or HCVpp produced in 293rVLP, 293rVLP-HCVpp, 293rVLP-HCVpp (AEE), 293rVLP-shCD81 cells and 293rVLP-HCVpp (BEE) cells determined by western blotting of purified particles; c surface displayed CD81 and HCV E2 proteins 293rVLP, 293rVLP-HCVpp, 293rVLP-HCVpp (AEE), 293rVLP-shCD81 cells and 293rVLP-HCVpp (BEE) cells determined by flow cytometry
Figure Legend Snippet: Cellular expression of and incorporation of HCV envelope proteins in chimeric HCVpp. a Cellular expression of MLV p30, CD81, HCV E1 and HCV E2 determined by western blotting in 293rVLP, 293rVLP-HCVpp, 293rVLP-HCVpp (AEE), 293rVLP-shCD81 cells and 293rVLP-HCVpp-shCD81 (BEE); b incorporation of CD81, HCV E1 and HCV E2 membrane proteins in null retrovirus particles or HCVpp produced in 293rVLP, 293rVLP-HCVpp, 293rVLP-HCVpp (AEE), 293rVLP-shCD81 cells and 293rVLP-HCVpp (BEE) cells determined by western blotting of purified particles; c surface displayed CD81 and HCV E2 proteins 293rVLP, 293rVLP-HCVpp, 293rVLP-HCVpp (AEE), 293rVLP-shCD81 cells and 293rVLP-HCVpp (BEE) cells determined by flow cytometry

Techniques Used: Expressing, Western Blot, Produced, Purification, Flow Cytometry, Cytometry

Validation of CD81-dependent transport of HCV E2 in HCV core expressing cells. a Schematic representation of the expression plasmids containing lentiviral vectors transgene sequences inducing the cellular expression of HCV Core, E1E2 and p7NS2 ORFs; b cellular expression of MLV p30, CD81, HCV E1 and HCV E2 determined by western blotting in HEK293, HEK293-HCV Core-NS2 , HEK293-HCV Core-NS2 shCD81, HuH-7, HuH7-HCV Core-NS2 and HuH7-HCV Core-NS2 shCD81. c Surface displayed CD81 and HCV E2 proteins in ( c ) HEK293, HEK293-HCV Core-NS2 , HEK293-HCV Core-NS2 shCD81 and d HuH-7, HuH7-HCV Core-NS2 and HuH7-HCV Core-NS2 shCD81
Figure Legend Snippet: Validation of CD81-dependent transport of HCV E2 in HCV core expressing cells. a Schematic representation of the expression plasmids containing lentiviral vectors transgene sequences inducing the cellular expression of HCV Core, E1E2 and p7NS2 ORFs; b cellular expression of MLV p30, CD81, HCV E1 and HCV E2 determined by western blotting in HEK293, HEK293-HCV Core-NS2 , HEK293-HCV Core-NS2 shCD81, HuH-7, HuH7-HCV Core-NS2 and HuH7-HCV Core-NS2 shCD81. c Surface displayed CD81 and HCV E2 proteins in ( c ) HEK293, HEK293-HCV Core-NS2 , HEK293-HCV Core-NS2 shCD81 and d HuH-7, HuH7-HCV Core-NS2 and HuH7-HCV Core-NS2 shCD81

Techniques Used: Expressing, Western Blot

Schematic representation of cell line development. 293rVLP cells producing non pseudotyped retrovirus like particles (Null VLPs) were transfected with HCV envelope E1 (green) and E2 (brown) proteins coding plasmid and produced HCV pseudoparticles (HCVpp), afterwards these cells were silenced for endogenous CD81 generating 293rVLP-HCVpp shCD81 (AEE—After Envelope Expression) (upper panel). In parallel, 293rVLP were silenced for endogenous CD81 expression to produce non-pseudotyped retrovirus like particles deprived from CD81, these cells were then transfected with a plasmid coding HCV envelope proteins generating 293rVLP-HCVpp shCD81 (BEE—Before Envelope Expression) (lower panel)
Figure Legend Snippet: Schematic representation of cell line development. 293rVLP cells producing non pseudotyped retrovirus like particles (Null VLPs) were transfected with HCV envelope E1 (green) and E2 (brown) proteins coding plasmid and produced HCV pseudoparticles (HCVpp), afterwards these cells were silenced for endogenous CD81 generating 293rVLP-HCVpp shCD81 (AEE—After Envelope Expression) (upper panel). In parallel, 293rVLP were silenced for endogenous CD81 expression to produce non-pseudotyped retrovirus like particles deprived from CD81, these cells were then transfected with a plasmid coding HCV envelope proteins generating 293rVLP-HCVpp shCD81 (BEE—Before Envelope Expression) (lower panel)

Techniques Used: Transfection, Plasmid Preparation, Produced, Expressing

10) Product Images from "Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *"

Article Title: Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.606269

EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized
Figure Legend Snippet: EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized

Techniques Used: Staining

CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells
Figure Legend Snippet: CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells

Techniques Used: Inhibition

11) Product Images from "CD81 association with SAMHD1 enhances HIV-1 reverse transcription by increasing dNTP levels"

Article Title: CD81 association with SAMHD1 enhances HIV-1 reverse transcription by increasing dNTP levels

Journal: Nature microbiology

doi: 10.1038/s41564-017-0019-0

CD81 regulates SAMHD1 expression. a) Primary T lymphoblasts transfected with control or CD81 siRNA, and Hela/R5 cells transfected or not with CRISPR/Cas9-CD81 were lysed and immunoblotted for CD81, SAMHD1 and phosphorylated SAMHD1. Tubulin or cofilin were used as loading controls. Blots are from representative experiments out of 2 (lymphoblasts) and 3 (Hela/R5). Signal ratios in relation to the loading controls are depicted. Arrows indicate the SAMHD1 band that has the predicted molecular weight (~70kDa). b) Hela/R5 cells transfected or not with CRISPR/Cas9-CD81 were fixed, permeabilized, immunolabelled for SAMHD1 and CD81, and analysed by flow cytometry. Histograms show a representative experiment out of 3. The negative control corresponds to cells stained only with secondary antibody. c-d) CRISPR/Cas9-CD81 or control Hela/R5 cells were treated for 6h with the vehicle or with the depicted concentrations of (c) MG132 or (d) NH 4 Cl. Cells were lysed and immunoblotted for SAMHD1, and ERM as loading control. Graph shows the mean fold change ± SEM of SAMHD1/ERM signal ratio from (c) 3 or (d) 2 independent experiments analysed by one-way ANOVA with Bonferroni’s post-test.
Figure Legend Snippet: CD81 regulates SAMHD1 expression. a) Primary T lymphoblasts transfected with control or CD81 siRNA, and Hela/R5 cells transfected or not with CRISPR/Cas9-CD81 were lysed and immunoblotted for CD81, SAMHD1 and phosphorylated SAMHD1. Tubulin or cofilin were used as loading controls. Blots are from representative experiments out of 2 (lymphoblasts) and 3 (Hela/R5). Signal ratios in relation to the loading controls are depicted. Arrows indicate the SAMHD1 band that has the predicted molecular weight (~70kDa). b) Hela/R5 cells transfected or not with CRISPR/Cas9-CD81 were fixed, permeabilized, immunolabelled for SAMHD1 and CD81, and analysed by flow cytometry. Histograms show a representative experiment out of 3. The negative control corresponds to cells stained only with secondary antibody. c-d) CRISPR/Cas9-CD81 or control Hela/R5 cells were treated for 6h with the vehicle or with the depicted concentrations of (c) MG132 or (d) NH 4 Cl. Cells were lysed and immunoblotted for SAMHD1, and ERM as loading control. Graph shows the mean fold change ± SEM of SAMHD1/ERM signal ratio from (c) 3 or (d) 2 independent experiments analysed by one-way ANOVA with Bonferroni’s post-test.

Techniques Used: Expressing, Transfection, CRISPR, Molecular Weight, Flow Cytometry, Cytometry, Negative Control, Staining

CD81 expression supports R5-tropic HIV-1 RT. a) Time course of HIV-1 RT measured by qPCR of early or late RT products at 24 or 48h post-infection. Hela/R5 expressing GFP, CD81GFP or CD81∆cytGFP were infected with HIV-1 (BaL) or HIV-VSV-G. Data are mean fold change ± SEM from 4 independent experiments performed in triplicate, and analysed by two-way ANOVA with Bonferroni’s post-test. b) Hela/R5 cells expressing GFP, CD81GFP or CD81∆cytGFP were infected with single-cycle HIV-1-R5-Luc or HIV-VSV-G-Luc. Data are mean fold-induction ± SEM of luciferase activity from 3 independent experiments performed in triplicate, and analysed by one-way ANOVA with Tukey’s post-test. c) Hela/R5 cells transfected with CRISPR/Cas9-CD81 or left untreated were infected and analysed as in b . Data are mean fold change ± SEM from 3 independent experiments performed in triplicate, and analysed by paired Student t -test, *** p
Figure Legend Snippet: CD81 expression supports R5-tropic HIV-1 RT. a) Time course of HIV-1 RT measured by qPCR of early or late RT products at 24 or 48h post-infection. Hela/R5 expressing GFP, CD81GFP or CD81∆cytGFP were infected with HIV-1 (BaL) or HIV-VSV-G. Data are mean fold change ± SEM from 4 independent experiments performed in triplicate, and analysed by two-way ANOVA with Bonferroni’s post-test. b) Hela/R5 cells expressing GFP, CD81GFP or CD81∆cytGFP were infected with single-cycle HIV-1-R5-Luc or HIV-VSV-G-Luc. Data are mean fold-induction ± SEM of luciferase activity from 3 independent experiments performed in triplicate, and analysed by one-way ANOVA with Tukey’s post-test. c) Hela/R5 cells transfected with CRISPR/Cas9-CD81 or left untreated were infected and analysed as in b . Data are mean fold change ± SEM from 3 independent experiments performed in triplicate, and analysed by paired Student t -test, *** p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Infection, Luciferase, Activity Assay, Transfection, CRISPR

CD81 regulates X4-tropic HIV-1 RT. a) Primary T lymphoblasts pre-treated with 2μM of scramble or CD81pept for 5 days, were infected with HIV-1 NL4-3 strain or HIV-VSV-G. Early or late RT products were measured as in Figure 2a . Data are mean fold change ± SEM of 2 independent experiments performed in triplicate, and analysed by two-way ANOVA with Bonferroni’s post-test. b) Primary T lymphoblasts transfected with control or CD81 siRNA were infected with NL4-3 strain, and RT was measured as in Figure 2a . Data are from a representative experiment out of two. In-box shows immunoblots of whole cell lysates from the represented experiment probed for CD81, and ERM as loading control. The CD81/ERM signal ratio is indicated.
Figure Legend Snippet: CD81 regulates X4-tropic HIV-1 RT. a) Primary T lymphoblasts pre-treated with 2μM of scramble or CD81pept for 5 days, were infected with HIV-1 NL4-3 strain or HIV-VSV-G. Early or late RT products were measured as in Figure 2a . Data are mean fold change ± SEM of 2 independent experiments performed in triplicate, and analysed by two-way ANOVA with Bonferroni’s post-test. b) Primary T lymphoblasts transfected with control or CD81 siRNA were infected with NL4-3 strain, and RT was measured as in Figure 2a . Data are from a representative experiment out of two. In-box shows immunoblots of whole cell lysates from the represented experiment probed for CD81, and ERM as loading control. The CD81/ERM signal ratio is indicated.

Techniques Used: Infection, Transfection, Western Blot

CD81 negatively regulates cellular dNTP content through SAMHD1. a) Jurkat T cells transfected with control or CD81 siRNA were infected with HIV-1 NL4-3 strain, and early RT products were measured by qPCR at the indicated times. Data are mean fold change ± SEM of 2 independent experiments performed in triplicate. In-box shows representative immunoblots of CD81 and tubulin as loading control, and the CD81/Tubulin signal ratio is indicated. b) Jurkat T cells pre-treated with 2μM of scramble or CD81pept for 5 days were infected with NL4-3 strain, and RT was analysed as in a . c) Mean fold change ± SEM of the dNTP content of Hela/R5 or Hela/R5 CRISPR/Cas9-CD81 cells (left graph, 4 independent experiments), and primary T lymphoblasts transfected with control or CD81 siRNA (right graph, 2 independent experiments) measured by a HIV RT-based dNTP assay. Analysis was performed by paired Student t -test, *** p
Figure Legend Snippet: CD81 negatively regulates cellular dNTP content through SAMHD1. a) Jurkat T cells transfected with control or CD81 siRNA were infected with HIV-1 NL4-3 strain, and early RT products were measured by qPCR at the indicated times. Data are mean fold change ± SEM of 2 independent experiments performed in triplicate. In-box shows representative immunoblots of CD81 and tubulin as loading control, and the CD81/Tubulin signal ratio is indicated. b) Jurkat T cells pre-treated with 2μM of scramble or CD81pept for 5 days were infected with NL4-3 strain, and RT was analysed as in a . c) Mean fold change ± SEM of the dNTP content of Hela/R5 or Hela/R5 CRISPR/Cas9-CD81 cells (left graph, 4 independent experiments), and primary T lymphoblasts transfected with control or CD81 siRNA (right graph, 2 independent experiments) measured by a HIV RT-based dNTP assay. Analysis was performed by paired Student t -test, *** p

Techniques Used: Transfection, Infection, Real-time Polymerase Chain Reaction, Western Blot, CRISPR

SAMHD1 is partially enriched at early endosomes. a) Hela/R5 cells were transfected with control or CD81 siRNA, adhered for 4h onto fibronectin (FN), fixed, permeabilized in PBS 0.1% Triton X-100 for 5min, and immunolabelled for SAMHD1. Images show one single confocal plane, nuclei are in blue. Arrows indicate SAMHD1 accumulation in circular-shaped intracellular structures, bars = 10μm. Graphs show means ± SEM of the number (counts/cell) and area (μm 2 /cell) of the cytoplasmic structures observed (n=230 cells, 4 independent experiments analysed by Student t -test, *** p = 0.0005 (upper) and *** p = 0.0006 (bottom)). b) Hela/R5 cells treated with 2μM of scramble or CD81pept were analysed as in a (n=400 cells, 4 independent experiments analysed by Student t -test, ** p = 0.0063 (upper) and ** p = 0.0054 (bottom)). c) Hela/R5 cells transfected with GFP, CD81GFP or CD81∆cytGFP were analysed as in a (n=20 cells, 2 independent experiments analysed by one-way ANOVA with Tukey’s post-test). d) Hela/R5 transfected with control or CD81 siRNA were treated as in a . Images show SAMHD1 (green), EEA1 (red), LAMP-1 (magenta), nuclei (blue), DIC, SAMHD1/EEA1 co-localization channel (white), and merged images. One single confocal plane is shown, bars = 10μm. Graphs represent the quantification of SAMHD1-EEA1 co-localization performed in 3D stack confocal microscopy images, showing means ± SEM of the Pearson’s coefficient; and of the % of SAMHD1 signal co-localized with EEA1 signal with respect to the total SAMHD1 signal in the cell (n = 200 cells, 3 independent experiments analysed by Student t -test, * p = 0.0262 (left) and * p = 0.0479 (right)).
Figure Legend Snippet: SAMHD1 is partially enriched at early endosomes. a) Hela/R5 cells were transfected with control or CD81 siRNA, adhered for 4h onto fibronectin (FN), fixed, permeabilized in PBS 0.1% Triton X-100 for 5min, and immunolabelled for SAMHD1. Images show one single confocal plane, nuclei are in blue. Arrows indicate SAMHD1 accumulation in circular-shaped intracellular structures, bars = 10μm. Graphs show means ± SEM of the number (counts/cell) and area (μm 2 /cell) of the cytoplasmic structures observed (n=230 cells, 4 independent experiments analysed by Student t -test, *** p = 0.0005 (upper) and *** p = 0.0006 (bottom)). b) Hela/R5 cells treated with 2μM of scramble or CD81pept were analysed as in a (n=400 cells, 4 independent experiments analysed by Student t -test, ** p = 0.0063 (upper) and ** p = 0.0054 (bottom)). c) Hela/R5 cells transfected with GFP, CD81GFP or CD81∆cytGFP were analysed as in a (n=20 cells, 2 independent experiments analysed by one-way ANOVA with Tukey’s post-test). d) Hela/R5 transfected with control or CD81 siRNA were treated as in a . Images show SAMHD1 (green), EEA1 (red), LAMP-1 (magenta), nuclei (blue), DIC, SAMHD1/EEA1 co-localization channel (white), and merged images. One single confocal plane is shown, bars = 10μm. Graphs represent the quantification of SAMHD1-EEA1 co-localization performed in 3D stack confocal microscopy images, showing means ± SEM of the Pearson’s coefficient; and of the % of SAMHD1 signal co-localized with EEA1 signal with respect to the total SAMHD1 signal in the cell (n = 200 cells, 3 independent experiments analysed by Student t -test, * p = 0.0262 (left) and * p = 0.0479 (right)).

Techniques Used: Transfection, Confocal Microscopy

The C-terminal domain of CD81 mediates its association with SAMHD1. a) SAMHD1 immunoblot of primary T lymphoblast lysates pulled-down with biotinylated peptides of tetraspanins CD81, CD9 and CD151 C-terminal domains. Sepharose-negative control and whole cell lysate are shown. b) Primary T lymphoblast lysates were immunoprecipitated and immunoblotted with SAMHD1 or CD81 antibodies. Control beads incubated with cell lysate, and whole cell lysate are shown. c) Hela/R5 cells or primary T lymphoblasts were plated onto PLL, fixed, permeabilized in PBS 0.5% Triton X-100 for 5 min, stained for CD81 (red) and SAMHD1 (green), and analysed by confocal microscopy. One single confocal plane is shown, nuclei are in blue. Bar = 10μm. d) Hela/R5 cells transfected with control siRNA (siControl) or CD81 siRNA (siCD81) were plated onto PLL (10μg/ml), anti-CD9 (VJ1/20, 10μg/ml), anti-CD4 (HP2/6, 10μg/ml) or anti-CD81 (5A6, 10μg/ml) monoclonal antibodies for 2h, fixed, permeabilized in 0.5% Triton X-100 for 5min, and stained for SAMHD1 (polyclonal antibody). Images show a single confocal plane at a ventral position, bar = 10μm. Graph shows means ± SEM of the number (counts/cell) of SAMHD1 + clusters (n=50 cells, 3 independent experiments), analysed by one-way ANOVA with Tukey’s post-test. e) Duo-link immunoassay of primary T lymphoblasts plated onto PLL, permeabilized in PBS 0.5% Triton X-100 for 5min, and stained for SAMHD1 and CD81. SAMHD1/CD147 and CD81/ERM were used as negative and positive controls respectively, bar = 10μm. Graph shows the number of dots per cell; each dot represents a single cell, bars denote the mean of scatter plots, and data was analysed by one-way ANOVA with Dunns post-test.
Figure Legend Snippet: The C-terminal domain of CD81 mediates its association with SAMHD1. a) SAMHD1 immunoblot of primary T lymphoblast lysates pulled-down with biotinylated peptides of tetraspanins CD81, CD9 and CD151 C-terminal domains. Sepharose-negative control and whole cell lysate are shown. b) Primary T lymphoblast lysates were immunoprecipitated and immunoblotted with SAMHD1 or CD81 antibodies. Control beads incubated with cell lysate, and whole cell lysate are shown. c) Hela/R5 cells or primary T lymphoblasts were plated onto PLL, fixed, permeabilized in PBS 0.5% Triton X-100 for 5 min, stained for CD81 (red) and SAMHD1 (green), and analysed by confocal microscopy. One single confocal plane is shown, nuclei are in blue. Bar = 10μm. d) Hela/R5 cells transfected with control siRNA (siControl) or CD81 siRNA (siCD81) were plated onto PLL (10μg/ml), anti-CD9 (VJ1/20, 10μg/ml), anti-CD4 (HP2/6, 10μg/ml) or anti-CD81 (5A6, 10μg/ml) monoclonal antibodies for 2h, fixed, permeabilized in 0.5% Triton X-100 for 5min, and stained for SAMHD1 (polyclonal antibody). Images show a single confocal plane at a ventral position, bar = 10μm. Graph shows means ± SEM of the number (counts/cell) of SAMHD1 + clusters (n=50 cells, 3 independent experiments), analysed by one-way ANOVA with Tukey’s post-test. e) Duo-link immunoassay of primary T lymphoblasts plated onto PLL, permeabilized in PBS 0.5% Triton X-100 for 5min, and stained for SAMHD1 and CD81. SAMHD1/CD147 and CD81/ERM were used as negative and positive controls respectively, bar = 10μm. Graph shows the number of dots per cell; each dot represents a single cell, bars denote the mean of scatter plots, and data was analysed by one-way ANOVA with Dunns post-test.

Techniques Used: Negative Control, Immunoprecipitation, Incubation, Staining, Confocal Microscopy, Transfection

12) Product Images from "Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *"

Article Title: Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.606269

EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized
Figure Legend Snippet: EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized

Techniques Used: Staining

CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells
Figure Legend Snippet: CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells

Techniques Used: Inhibition

13) Product Images from "Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *"

Article Title: Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.606269

EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized
Figure Legend Snippet: EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized

Techniques Used: Staining

CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells
Figure Legend Snippet: CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells

Techniques Used: Inhibition

14) Product Images from "Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *"

Article Title: Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.606269

EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized
Figure Legend Snippet: EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized

Techniques Used: Staining

CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells
Figure Legend Snippet: CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells

Techniques Used: Inhibition

15) Product Images from "Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes"

Article Title: Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes

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

doi: 10.1073/pnas.1521230113

Qualitative and quantitative proteomic analyses of sEVs immuno-isolated by CD9, CD63, or CD81-specific antibodies evidence additional sEV subpopulations. ( A ) The crude DC-derived 100K pellet was subjected to parallel immuno-isolation with beads coupled
Figure Legend Snippet: Qualitative and quantitative proteomic analyses of sEVs immuno-isolated by CD9, CD63, or CD81-specific antibodies evidence additional sEV subpopulations. ( A ) The crude DC-derived 100K pellet was subjected to parallel immuno-isolation with beads coupled

Techniques Used: Isolation, Derivative Assay

16) Product Images from "Characterization of plasma circulating small extracellular vesicles in patients with metastatic solid tumors and newly diagnosed brain metastasis"

Article Title: Characterization of plasma circulating small extracellular vesicles in patients with metastatic solid tumors and newly diagnosed brain metastasis

Journal: Oncoimmunology

doi: 10.1080/2162402X.2022.2067944

Overall survival analysis of patients included in the study and characterization of plasma-circulating sEVs. a . Survival analysis and graphical representation using Kaplan-Meier curves showing the cumulative survival probability in the study population according to the absence (CNS-) or presence (CNS+) of central nervous system (CNS) metastases. * p value 0.02. Differences were assessed using the Log-Rank test. b . Representative image of the particle content (x10 8 ) by NTA analysis of a plasma sample from a melanoma patient. c . Representative electron microscopy imaging of sEVs from the same patient´s plasma. d . Representative Western blot of the analysis of exosome markers CD9, CD81, TSG101 and in sEVs isolated from the plasma of three different melanoma patients. Ponceau staining was used as loading control (see Supplementary Figure 4A). e . Proteomic analysis of sEVs and plasma paired samples derived from melanoma patients. Venn diagrams showing an enrichment of Exocarta markers (green) and a reduction albumin and apoliproteins (red) in sEVs (left diagram) compared to proteins detected in plasma samples (right diagram).
Figure Legend Snippet: Overall survival analysis of patients included in the study and characterization of plasma-circulating sEVs. a . Survival analysis and graphical representation using Kaplan-Meier curves showing the cumulative survival probability in the study population according to the absence (CNS-) or presence (CNS+) of central nervous system (CNS) metastases. * p value 0.02. Differences were assessed using the Log-Rank test. b . Representative image of the particle content (x10 8 ) by NTA analysis of a plasma sample from a melanoma patient. c . Representative electron microscopy imaging of sEVs from the same patient´s plasma. d . Representative Western blot of the analysis of exosome markers CD9, CD81, TSG101 and in sEVs isolated from the plasma of three different melanoma patients. Ponceau staining was used as loading control (see Supplementary Figure 4A). e . Proteomic analysis of sEVs and plasma paired samples derived from melanoma patients. Venn diagrams showing an enrichment of Exocarta markers (green) and a reduction albumin and apoliproteins (red) in sEVs (left diagram) compared to proteins detected in plasma samples (right diagram).

Techniques Used: Electron Microscopy, Imaging, Western Blot, Isolation, Staining, Derivative Assay

17) Product Images from "Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *"

Article Title: Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.606269

EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized
Figure Legend Snippet: EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized

Techniques Used: Staining

CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells
Figure Legend Snippet: CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells

Techniques Used: Inhibition

18) Product Images from "Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *"

Article Title: Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.606269

EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized
Figure Legend Snippet: EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized

Techniques Used: Staining

CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells
Figure Legend Snippet: CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells

Techniques Used: Inhibition

19) Product Images from "Exosomes Released From Human Bone Marrow–Derived Mesenchymal Stem Cell Attenuate Acute Graft-Versus-Host Disease After Allogeneic Hematopoietic Stem Cell Transplantation in Mice"

Article Title: Exosomes Released From Human Bone Marrow–Derived Mesenchymal Stem Cell Attenuate Acute Graft-Versus-Host Disease After Allogeneic Hematopoietic Stem Cell Transplantation in Mice

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2021.617589

Characteristics of human bone marrow–derived mesenchymal stem cell (hBMSC)–derived exosomes. (A) Representative images of hBMSC-derived exosomes under transmission electron microscopy. Scale bar: 500 and 200 nm. (B) Size distributions of hBMSC-derived exosomes determined by dynamic light scattering. (C) Positive expression of CD9, CD63, and CD81 in isolated exosomes by western blot. β-Actin was used as the control for western blot. Fib-lys, the total cell lysate of human fibroblasts; Fib-exo, the exosomes from human fibroblasts; Msc-exo, the exosomes from human bone marrow–derived mesenchymal stem cells.
Figure Legend Snippet: Characteristics of human bone marrow–derived mesenchymal stem cell (hBMSC)–derived exosomes. (A) Representative images of hBMSC-derived exosomes under transmission electron microscopy. Scale bar: 500 and 200 nm. (B) Size distributions of hBMSC-derived exosomes determined by dynamic light scattering. (C) Positive expression of CD9, CD63, and CD81 in isolated exosomes by western blot. β-Actin was used as the control for western blot. Fib-lys, the total cell lysate of human fibroblasts; Fib-exo, the exosomes from human fibroblasts; Msc-exo, the exosomes from human bone marrow–derived mesenchymal stem cells.

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

20) Product Images from "Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *"

Article Title: Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.606269

EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized
Figure Legend Snippet: EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized

Techniques Used: Staining

CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells
Figure Legend Snippet: CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells

Techniques Used: Inhibition

21) Product Images from "Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *"

Article Title: Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.606269

EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized
Figure Legend Snippet: EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized

Techniques Used: Staining

CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells
Figure Legend Snippet: CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells

Techniques Used: Inhibition

22) Product Images from "MicroRNA and Protein Profiling of Brain Metastasis Competent Cell-Derived Exosomes"

Article Title: MicroRNA and Protein Profiling of Brain Metastasis Competent Cell-Derived Exosomes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0073790

Identification and characterization of isolated exosomes. Exosomes were isolated by the ExoQuick-TC Precipitation Solution. ( A ) Representative morphological characterization of exosomes derived from brain metastatic 70 W melanoma cells by transmission electron microscopy. Round particles with characteristic exosomal size (30–100 nm) and shape were observed (arrows) immersed in the Exoquick solution. Scale bar is 50 nm. ( B ) Molecular confirmation of exosomes markers by Western blotting analysis. Exosome preparations were found to be positive for the exosomal markers CD9, CD63, and CD81 while negative for proteins from the endoplasmic reticulum (calnexin) and the Golgi apparatus (GM130) which were found to be present in cells lysates.
Figure Legend Snippet: Identification and characterization of isolated exosomes. Exosomes were isolated by the ExoQuick-TC Precipitation Solution. ( A ) Representative morphological characterization of exosomes derived from brain metastatic 70 W melanoma cells by transmission electron microscopy. Round particles with characteristic exosomal size (30–100 nm) and shape were observed (arrows) immersed in the Exoquick solution. Scale bar is 50 nm. ( B ) Molecular confirmation of exosomes markers by Western blotting analysis. Exosome preparations were found to be positive for the exosomal markers CD9, CD63, and CD81 while negative for proteins from the endoplasmic reticulum (calnexin) and the Golgi apparatus (GM130) which were found to be present in cells lysates.

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

23) Product Images from "Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *"

Article Title: Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.606269

EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized
Figure Legend Snippet: EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized

Techniques Used: Staining

CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells
Figure Legend Snippet: CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells

Techniques Used: Inhibition

24) Product Images from "Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *"

Article Title: Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.606269

EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized
Figure Legend Snippet: EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized

Techniques Used: Staining

CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells
Figure Legend Snippet: CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells

Techniques Used: Inhibition

25) Product Images from "Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *"

Article Title: Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.606269

EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized
Figure Legend Snippet: EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized

Techniques Used: Staining

CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells
Figure Legend Snippet: CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells

Techniques Used: Inhibition

26) Product Images from "Interferon-α inducible protein 6 impairs EGFR activation by CD81 and inhibits hepatitis C virus infection"

Article Title: Interferon-α inducible protein 6 impairs EGFR activation by CD81 and inhibits hepatitis C virus infection

Journal: Scientific Reports

doi: 10.1038/srep09012

IFI6 expression inhibits CD81 and CLDN1 interactions during antibody cross-linked activation of CD81. Expression of CD81 and CLDN1 on the cell surface of unstimulated control and Huh7.5 stable transfectants expressing IFI6 is shown (panels A and E; respectively). CD81 (panel B) and CLDN1 (panel C) were detected in fixed mock-transfected control Huh7.5 cells. Merged fluorescence showing translocation of CD81 and co-localization with CLDN1 in tight junctions of cells (panel D, 56% co-localization of claudin with CD81 per field). CD81 (panel F), CLDN1 (panel G), and merged immunofluorescence analysis displaying localization (panel H,
Figure Legend Snippet: IFI6 expression inhibits CD81 and CLDN1 interactions during antibody cross-linked activation of CD81. Expression of CD81 and CLDN1 on the cell surface of unstimulated control and Huh7.5 stable transfectants expressing IFI6 is shown (panels A and E; respectively). CD81 (panel B) and CLDN1 (panel C) were detected in fixed mock-transfected control Huh7.5 cells. Merged fluorescence showing translocation of CD81 and co-localization with CLDN1 in tight junctions of cells (panel D, 56% co-localization of claudin with CD81 per field). CD81 (panel F), CLDN1 (panel G), and merged immunofluorescence analysis displaying localization (panel H,

Techniques Used: Expressing, Activation Assay, Transfection, Fluorescence, Translocation Assay, Immunofluorescence

IFI6 localizes on both the cell surface and intracellularly, and does not associate with CD81. FLAG tagged IFI6 expression was detected in lysates of Huh7.5 stable transfectants by Western blotting with a FLAG specific antibody. Note that cropped gel images are used in this figure and the gels were run under the same experimental conditions (panel A). Expression of IFI6 is shown during transient, stable, and IFN-α stimulated conditions; and standard deviations are shown as error bars (panel B). Detection of IFI6 on the cell surface of unfixed (panel C, section a) and in the cytoplasm of fixed cells (panel C, section b) by FLAG antibody. Mitochondria were stained with a mitotracker dye (panel C, section c) and merged fluorescence of cytoplasmic IFI6 and stained mitochondria (panel C, section d) are shown. Basal expression of CD81 was determined in Huh7.5 cells (panel D, Section a) and IFI6 transfected Huh7.5 cells (panel D, section b). Huh7.5 and IFI6 expressing cells were cross-linked with a CD81 specific antibody, followed by fixation. CD81 translocation after antibody mediated cross-linking was observed in control (panel D, section c) and was not inhibited in IFI6 expressing cells (panel D, section d). The presence of IFI6 (red) was also observed (panel C, section D) and did not co-localize with CD81 (green). Cell nuclei were stained with DAPI (blue). Each panel is representative of three independent experiments.
Figure Legend Snippet: IFI6 localizes on both the cell surface and intracellularly, and does not associate with CD81. FLAG tagged IFI6 expression was detected in lysates of Huh7.5 stable transfectants by Western blotting with a FLAG specific antibody. Note that cropped gel images are used in this figure and the gels were run under the same experimental conditions (panel A). Expression of IFI6 is shown during transient, stable, and IFN-α stimulated conditions; and standard deviations are shown as error bars (panel B). Detection of IFI6 on the cell surface of unfixed (panel C, section a) and in the cytoplasm of fixed cells (panel C, section b) by FLAG antibody. Mitochondria were stained with a mitotracker dye (panel C, section c) and merged fluorescence of cytoplasmic IFI6 and stained mitochondria (panel C, section d) are shown. Basal expression of CD81 was determined in Huh7.5 cells (panel D, Section a) and IFI6 transfected Huh7.5 cells (panel D, section b). Huh7.5 and IFI6 expressing cells were cross-linked with a CD81 specific antibody, followed by fixation. CD81 translocation after antibody mediated cross-linking was observed in control (panel D, section c) and was not inhibited in IFI6 expressing cells (panel D, section d). The presence of IFI6 (red) was also observed (panel C, section D) and did not co-localize with CD81 (green). Cell nuclei were stained with DAPI (blue). Each panel is representative of three independent experiments.

Techniques Used: Expressing, Western Blot, Staining, Fluorescence, Transfection, Translocation Assay

IFI6 expression inhibits EGFR activation in the presence of CD81 antibody. IFI6 stably transfected cells were treated with EGF and cell lysates were analyzed for phosphorylated EGFR. Expression of IFI6 inhibited EGFR activation by CD81 cross-linking, but not from EGF treatment (panel A). Cells stimulated with antibody to CD81 were lysed in a reducing sample buffer and subjected to Western blot analysis for EGFR activation (panel B). Blots were reprobed with antibody to actin as a loading control. Quantification of protein levels was performed using ImageJ software from three separate experiments and standard deviations are shown as error bars. Note that cropped gel images are used in this figure and the gels were run under the same experimental conditions.
Figure Legend Snippet: IFI6 expression inhibits EGFR activation in the presence of CD81 antibody. IFI6 stably transfected cells were treated with EGF and cell lysates were analyzed for phosphorylated EGFR. Expression of IFI6 inhibited EGFR activation by CD81 cross-linking, but not from EGF treatment (panel A). Cells stimulated with antibody to CD81 were lysed in a reducing sample buffer and subjected to Western blot analysis for EGFR activation (panel B). Blots were reprobed with antibody to actin as a loading control. Quantification of protein levels was performed using ImageJ software from three separate experiments and standard deviations are shown as error bars. Note that cropped gel images are used in this figure and the gels were run under the same experimental conditions.

Techniques Used: Expressing, Activation Assay, Stable Transfection, Transfection, Western Blot, Software

IFI6 expression inhibits CD81 and CLDN1 interactions during HCV infection. Huh7.5 control and IFI6 expressing cells were infected with HCVcc and co-localization of CD81 and CLDN1 was determined. Expression of CD81 and CLDN1 on the cell surface of unstimulated control and IFI6 expressing cells is shown (panel A and E, respectively). CD81 (panel B) and CLDN1 (panel C) were detected in fixed mock-transfected control cells. Merged immunofluorescence showing translocation of CD81 and co-localization with CLDN1 in tight junctions of cells is shown (panel D, 6.1% co-localization of CLDN1 with CD81 per field). CD81 (panel F), CLDN1 (panel G), and merged fluorescence displaying localization (panel H, 0.2% co-localization of CLDN1 with CD81 per field) are shown in IFI6 transfected cells. Each panel is representative of three independent experiments.
Figure Legend Snippet: IFI6 expression inhibits CD81 and CLDN1 interactions during HCV infection. Huh7.5 control and IFI6 expressing cells were infected with HCVcc and co-localization of CD81 and CLDN1 was determined. Expression of CD81 and CLDN1 on the cell surface of unstimulated control and IFI6 expressing cells is shown (panel A and E, respectively). CD81 (panel B) and CLDN1 (panel C) were detected in fixed mock-transfected control cells. Merged immunofluorescence showing translocation of CD81 and co-localization with CLDN1 in tight junctions of cells is shown (panel D, 6.1% co-localization of CLDN1 with CD81 per field). CD81 (panel F), CLDN1 (panel G), and merged fluorescence displaying localization (panel H, 0.2% co-localization of CLDN1 with CD81 per field) are shown in IFI6 transfected cells. Each panel is representative of three independent experiments.

Techniques Used: Expressing, Infection, Transfection, Immunofluorescence, Translocation Assay, Fluorescence

Activation of Ras/Raf/Erk pathway is inhibited in IFI6 expressing cells. Huh7.5 cells treated with antibody to CD81 for the indicated time were lysed in a reducing sample buffer and subjected to Western blot analysis. Lysates were probed for phosphorylated Raf-1 (Ser259). In addition, IFI6 expressing cells were stimulated with EGF and compared to antibody CD81 cross-linked cells to detect the loss of phosphorylation of Raf-1 at Ser259 (panel A). Activation of Raf-1 was analyzed by measuring phosphorylation at Ser338 cross-linking of CD81 or treatment with EGF (panel B). Blots were reprobed with antibody to actin as a loading control. Quantification of protein levels was performed using ImageJ software from three separate experiments and standard deviations are shown as error bars. Note that cropped gel images are used in this figure and the gels were run under the same experimental conditions.
Figure Legend Snippet: Activation of Ras/Raf/Erk pathway is inhibited in IFI6 expressing cells. Huh7.5 cells treated with antibody to CD81 for the indicated time were lysed in a reducing sample buffer and subjected to Western blot analysis. Lysates were probed for phosphorylated Raf-1 (Ser259). In addition, IFI6 expressing cells were stimulated with EGF and compared to antibody CD81 cross-linked cells to detect the loss of phosphorylation of Raf-1 at Ser259 (panel A). Activation of Raf-1 was analyzed by measuring phosphorylation at Ser338 cross-linking of CD81 or treatment with EGF (panel B). Blots were reprobed with antibody to actin as a loading control. Quantification of protein levels was performed using ImageJ software from three separate experiments and standard deviations are shown as error bars. Note that cropped gel images are used in this figure and the gels were run under the same experimental conditions.

Techniques Used: Activation Assay, Expressing, Western Blot, Software

27) Product Images from "Interferon-α inducible protein 6 impairs EGFR activation by CD81 and inhibits hepatitis C virus infection"

Article Title: Interferon-α inducible protein 6 impairs EGFR activation by CD81 and inhibits hepatitis C virus infection

Journal: Scientific Reports

doi: 10.1038/srep09012

IFI6 expression inhibits CD81 and CLDN1 interactions during antibody cross-linked activation of CD81. Expression of CD81 and CLDN1 on the cell surface of unstimulated control and Huh7.5 stable transfectants expressing IFI6 is shown (panels A and E; respectively). CD81 (panel B) and CLDN1 (panel C) were detected in fixed mock-transfected control Huh7.5 cells. Merged fluorescence showing translocation of CD81 and co-localization with CLDN1 in tight junctions of cells (panel D, 56% co-localization of claudin with CD81 per field). CD81 (panel F), CLDN1 (panel G), and merged immunofluorescence analysis displaying localization (panel H,
Figure Legend Snippet: IFI6 expression inhibits CD81 and CLDN1 interactions during antibody cross-linked activation of CD81. Expression of CD81 and CLDN1 on the cell surface of unstimulated control and Huh7.5 stable transfectants expressing IFI6 is shown (panels A and E; respectively). CD81 (panel B) and CLDN1 (panel C) were detected in fixed mock-transfected control Huh7.5 cells. Merged fluorescence showing translocation of CD81 and co-localization with CLDN1 in tight junctions of cells (panel D, 56% co-localization of claudin with CD81 per field). CD81 (panel F), CLDN1 (panel G), and merged immunofluorescence analysis displaying localization (panel H,

Techniques Used: Expressing, Activation Assay, Transfection, Fluorescence, Translocation Assay, Immunofluorescence

IFI6 localizes on both the cell surface and intracellularly, and does not associate with CD81. FLAG tagged IFI6 expression was detected in lysates of Huh7.5 stable transfectants by Western blotting with a FLAG specific antibody. Note that cropped gel images are used in this figure and the gels were run under the same experimental conditions (panel A). Expression of IFI6 is shown during transient, stable, and IFN-α stimulated conditions; and standard deviations are shown as error bars (panel B). Detection of IFI6 on the cell surface of unfixed (panel C, section a) and in the cytoplasm of fixed cells (panel C, section b) by FLAG antibody. Mitochondria were stained with a mitotracker dye (panel C, section c) and merged fluorescence of cytoplasmic IFI6 and stained mitochondria (panel C, section d) are shown. Basal expression of CD81 was determined in Huh7.5 cells (panel D, Section a) and IFI6 transfected Huh7.5 cells (panel D, section b). Huh7.5 and IFI6 expressing cells were cross-linked with a CD81 specific antibody, followed by fixation. CD81 translocation after antibody mediated cross-linking was observed in control (panel D, section c) and was not inhibited in IFI6 expressing cells (panel D, section d). The presence of IFI6 (red) was also observed (panel C, section D) and did not co-localize with CD81 (green). Cell nuclei were stained with DAPI (blue). Each panel is representative of three independent experiments.
Figure Legend Snippet: IFI6 localizes on both the cell surface and intracellularly, and does not associate with CD81. FLAG tagged IFI6 expression was detected in lysates of Huh7.5 stable transfectants by Western blotting with a FLAG specific antibody. Note that cropped gel images are used in this figure and the gels were run under the same experimental conditions (panel A). Expression of IFI6 is shown during transient, stable, and IFN-α stimulated conditions; and standard deviations are shown as error bars (panel B). Detection of IFI6 on the cell surface of unfixed (panel C, section a) and in the cytoplasm of fixed cells (panel C, section b) by FLAG antibody. Mitochondria were stained with a mitotracker dye (panel C, section c) and merged fluorescence of cytoplasmic IFI6 and stained mitochondria (panel C, section d) are shown. Basal expression of CD81 was determined in Huh7.5 cells (panel D, Section a) and IFI6 transfected Huh7.5 cells (panel D, section b). Huh7.5 and IFI6 expressing cells were cross-linked with a CD81 specific antibody, followed by fixation. CD81 translocation after antibody mediated cross-linking was observed in control (panel D, section c) and was not inhibited in IFI6 expressing cells (panel D, section d). The presence of IFI6 (red) was also observed (panel C, section D) and did not co-localize with CD81 (green). Cell nuclei were stained with DAPI (blue). Each panel is representative of three independent experiments.

Techniques Used: Expressing, Western Blot, Staining, Fluorescence, Transfection, Translocation Assay

IFI6 expression inhibits EGFR activation in the presence of CD81 antibody. IFI6 stably transfected cells were treated with EGF and cell lysates were analyzed for phosphorylated EGFR. Expression of IFI6 inhibited EGFR activation by CD81 cross-linking, but not from EGF treatment (panel A). Cells stimulated with antibody to CD81 were lysed in a reducing sample buffer and subjected to Western blot analysis for EGFR activation (panel B). Blots were reprobed with antibody to actin as a loading control. Quantification of protein levels was performed using ImageJ software from three separate experiments and standard deviations are shown as error bars. Note that cropped gel images are used in this figure and the gels were run under the same experimental conditions.
Figure Legend Snippet: IFI6 expression inhibits EGFR activation in the presence of CD81 antibody. IFI6 stably transfected cells were treated with EGF and cell lysates were analyzed for phosphorylated EGFR. Expression of IFI6 inhibited EGFR activation by CD81 cross-linking, but not from EGF treatment (panel A). Cells stimulated with antibody to CD81 were lysed in a reducing sample buffer and subjected to Western blot analysis for EGFR activation (panel B). Blots were reprobed with antibody to actin as a loading control. Quantification of protein levels was performed using ImageJ software from three separate experiments and standard deviations are shown as error bars. Note that cropped gel images are used in this figure and the gels were run under the same experimental conditions.

Techniques Used: Expressing, Activation Assay, Stable Transfection, Transfection, Western Blot, Software

IFI6 expression inhibits CD81 and CLDN1 interactions during HCV infection. Huh7.5 control and IFI6 expressing cells were infected with HCVcc and co-localization of CD81 and CLDN1 was determined. Expression of CD81 and CLDN1 on the cell surface of unstimulated control and IFI6 expressing cells is shown (panel A and E, respectively). CD81 (panel B) and CLDN1 (panel C) were detected in fixed mock-transfected control cells. Merged immunofluorescence showing translocation of CD81 and co-localization with CLDN1 in tight junctions of cells is shown (panel D, 6.1% co-localization of CLDN1 with CD81 per field). CD81 (panel F), CLDN1 (panel G), and merged fluorescence displaying localization (panel H, 0.2% co-localization of CLDN1 with CD81 per field) are shown in IFI6 transfected cells. Each panel is representative of three independent experiments.
Figure Legend Snippet: IFI6 expression inhibits CD81 and CLDN1 interactions during HCV infection. Huh7.5 control and IFI6 expressing cells were infected with HCVcc and co-localization of CD81 and CLDN1 was determined. Expression of CD81 and CLDN1 on the cell surface of unstimulated control and IFI6 expressing cells is shown (panel A and E, respectively). CD81 (panel B) and CLDN1 (panel C) were detected in fixed mock-transfected control cells. Merged immunofluorescence showing translocation of CD81 and co-localization with CLDN1 in tight junctions of cells is shown (panel D, 6.1% co-localization of CLDN1 with CD81 per field). CD81 (panel F), CLDN1 (panel G), and merged fluorescence displaying localization (panel H, 0.2% co-localization of CLDN1 with CD81 per field) are shown in IFI6 transfected cells. Each panel is representative of three independent experiments.

Techniques Used: Expressing, Infection, Transfection, Immunofluorescence, Translocation Assay, Fluorescence

Activation of Ras/Raf/Erk pathway is inhibited in IFI6 expressing cells. Huh7.5 cells treated with antibody to CD81 for the indicated time were lysed in a reducing sample buffer and subjected to Western blot analysis. Lysates were probed for phosphorylated Raf-1 (Ser259). In addition, IFI6 expressing cells were stimulated with EGF and compared to antibody CD81 cross-linked cells to detect the loss of phosphorylation of Raf-1 at Ser259 (panel A). Activation of Raf-1 was analyzed by measuring phosphorylation at Ser338 cross-linking of CD81 or treatment with EGF (panel B). Blots were reprobed with antibody to actin as a loading control. Quantification of protein levels was performed using ImageJ software from three separate experiments and standard deviations are shown as error bars. Note that cropped gel images are used in this figure and the gels were run under the same experimental conditions.
Figure Legend Snippet: Activation of Ras/Raf/Erk pathway is inhibited in IFI6 expressing cells. Huh7.5 cells treated with antibody to CD81 for the indicated time were lysed in a reducing sample buffer and subjected to Western blot analysis. Lysates were probed for phosphorylated Raf-1 (Ser259). In addition, IFI6 expressing cells were stimulated with EGF and compared to antibody CD81 cross-linked cells to detect the loss of phosphorylation of Raf-1 at Ser259 (panel A). Activation of Raf-1 was analyzed by measuring phosphorylation at Ser338 cross-linking of CD81 or treatment with EGF (panel B). Blots were reprobed with antibody to actin as a loading control. Quantification of protein levels was performed using ImageJ software from three separate experiments and standard deviations are shown as error bars. Note that cropped gel images are used in this figure and the gels were run under the same experimental conditions.

Techniques Used: Activation Assay, Expressing, Western Blot, Software

28) Product Images from "Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *"

Article Title: Complex N-Linked Glycans Serve as a Determinant for Exosome/Microvesicle Cargo Recruitment *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.606269

EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized
Figure Legend Snippet: EMV markers and complex N- glycans colocalize in discrete cell surface domains. A , fixed Sk-Mel-5 cells (4% PFA) were co-stained with DSA (biotin-DSA, Cy5-α-biotin mAb, red ) and CD81 antibody (FITC-α-CD81, green ). The cells were not permeabilized

Techniques Used: Staining

CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells
Figure Legend Snippet: CD81 recruitment to EMV is glycan-dependent but not EWI-2-dependent. A , inhibition of complex N- glycan alters CD81 trafficking to EMV. Sk-Mel-5 cells were treated with DMJ (1 m m ) for 48 h before collection of TCM or EMV (+ DMJ ). Untreated Sk-Mel-5 cells

Techniques Used: Inhibition

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    Millipore cd81 protein
    Size exclusion chromatography (SEC) analysis of <t>CD81-SMALP</t> reveals two distinct protein populations. A) Following Ni-NTA affinity chromatography, elution fractions were pooled, concentrated and 500 μl at 1 mg/ml were loaded on a Superdex 200 30/10 size exclusion column at 0.5 ml/min. Protein absorbance was monitored at 280 nm. B C) Fractions from the two main peaks obtained by SEC were run on SDS-PAGE and stained with Instant Blue. D) Fractions from the two SEC peaks were analysed for binding to the E2 glycoprotein (that acts as CD81 ligand) by ELISA, n = 2.
    Cd81 Protein, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cd81 protein/product/Millipore
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    98
    Millipore cav 1 cd8 cells
    Caveolin-1 promotes Ag-induced membrane raft synaptic polarization and actin polymerization in <t>CD8</t> + T cells. A, Left panel , Phase and fluorescent microscopy of raft-associated GM1 glycolipid localization in CD8 + cells from OT-1 <t>cav-1</t> +/+ and cav-1 −/−
    Cav 1 Cd8 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Size exclusion chromatography (SEC) analysis of CD81-SMALP reveals two distinct protein populations. A) Following Ni-NTA affinity chromatography, elution fractions were pooled, concentrated and 500 μl at 1 mg/ml were loaded on a Superdex 200 30/10 size exclusion column at 0.5 ml/min. Protein absorbance was monitored at 280 nm. B C) Fractions from the two main peaks obtained by SEC were run on SDS-PAGE and stained with Instant Blue. D) Fractions from the two SEC peaks were analysed for binding to the E2 glycoprotein (that acts as CD81 ligand) by ELISA, n = 2.

    Journal: Biochimica et Biophysica Acta. Biomembranes

    Article Title: CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups

    doi: 10.1016/j.bbamem.2020.183419

    Figure Lengend Snippet: Size exclusion chromatography (SEC) analysis of CD81-SMALP reveals two distinct protein populations. A) Following Ni-NTA affinity chromatography, elution fractions were pooled, concentrated and 500 μl at 1 mg/ml were loaded on a Superdex 200 30/10 size exclusion column at 0.5 ml/min. Protein absorbance was monitored at 280 nm. B C) Fractions from the two main peaks obtained by SEC were run on SDS-PAGE and stained with Instant Blue. D) Fractions from the two SEC peaks were analysed for binding to the E2 glycoprotein (that acts as CD81 ligand) by ELISA, n = 2.

    Article Snippet: CD81 protein samples were bound to plates, blocked and washed as described above.

    Techniques: Size-exclusion Chromatography, Affinity Chromatography, SDS Page, Staining, Binding Assay, Enzyme-linked Immunosorbent Assay

    Negative ion mode MS spectra measured for total lipid extracts of total yeast membrane, SMA solubilised membrane and purified CD81 SMALP. Identified glycerophosphatidylcholines are labelled in green (PC), glycerophosphatidyletanolamines in blue (PE), glycerophosphatidylinositols in magenta (PI), glycerophosphatidylserines in orange (PS) and cardiolipins in red (CL). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Journal: Biochimica et Biophysica Acta. Biomembranes

    Article Title: CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups

    doi: 10.1016/j.bbamem.2020.183419

    Figure Lengend Snippet: Negative ion mode MS spectra measured for total lipid extracts of total yeast membrane, SMA solubilised membrane and purified CD81 SMALP. Identified glycerophosphatidylcholines are labelled in green (PC), glycerophosphatidyletanolamines in blue (PE), glycerophosphatidylinositols in magenta (PI), glycerophosphatidylserines in orange (PS) and cardiolipins in red (CL). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: CD81 protein samples were bound to plates, blocked and washed as described above.

    Techniques: Purification

    Optimization of SEC conditions increases the proportion of E2-binding-competent CD81. A) SEC profile of purified CD81-SMALP obtained using standard conditions. B) SDS-PAGE of the CD81 sample loaded on the column, peak 1 and peak 2 from the SEC trace in A. C) SEC profile of purified CD81-SMALP obtained using modified expression and buffer conditions. D) SDS-PAGE of the CD81 sample loaded on the column, peak 1 and peak 2 from the SEC trace in C. Traces are representative of those seen in 3 repeats.

    Journal: Biochimica et Biophysica Acta. Biomembranes

    Article Title: CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups

    doi: 10.1016/j.bbamem.2020.183419

    Figure Lengend Snippet: Optimization of SEC conditions increases the proportion of E2-binding-competent CD81. A) SEC profile of purified CD81-SMALP obtained using standard conditions. B) SDS-PAGE of the CD81 sample loaded on the column, peak 1 and peak 2 from the SEC trace in A. C) SEC profile of purified CD81-SMALP obtained using modified expression and buffer conditions. D) SDS-PAGE of the CD81 sample loaded on the column, peak 1 and peak 2 from the SEC trace in C. Traces are representative of those seen in 3 repeats.

    Article Snippet: CD81 protein samples were bound to plates, blocked and washed as described above.

    Techniques: Binding Assay, Purification, SDS Page, Modification, Expressing

    Thermostability of purified CD81. Effect of temperature on secondary structure measured by CD spectroscopy over a range of different temperatures for A) CD81-SMALP and B) CD81-DDM. Temperatures ranged from 25 °C to 90 °Cin 5 °C increments. Eighteen technical replicates were taken at each condition, n = 2. C) The temperature dependenceof the CD signal at 207 nm for both CD81-SMALP (black closed circles) and CD81-DDM (grey open circles). D E) Effect of temperature on protein aggregation analysed by Western blot using an anti-CD81 antibody (mAb 2s131) for D) CD81-SMALP and E) CD81-DDM. F) Effect of increasing temperature on the binding of CD81 to a conformation sensitive anti-CD81 (mAb 1s337) antibody, using an ELISA. Black solid circles are CD81-SMALP, open grey squares are CD81-DDM. Data are mean ± SEM, n = 3.

    Journal: Biochimica et Biophysica Acta. Biomembranes

    Article Title: CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups

    doi: 10.1016/j.bbamem.2020.183419

    Figure Lengend Snippet: Thermostability of purified CD81. Effect of temperature on secondary structure measured by CD spectroscopy over a range of different temperatures for A) CD81-SMALP and B) CD81-DDM. Temperatures ranged from 25 °C to 90 °Cin 5 °C increments. Eighteen technical replicates were taken at each condition, n = 2. C) The temperature dependenceof the CD signal at 207 nm for both CD81-SMALP (black closed circles) and CD81-DDM (grey open circles). D E) Effect of temperature on protein aggregation analysed by Western blot using an anti-CD81 antibody (mAb 2s131) for D) CD81-SMALP and E) CD81-DDM. F) Effect of increasing temperature on the binding of CD81 to a conformation sensitive anti-CD81 (mAb 1s337) antibody, using an ELISA. Black solid circles are CD81-SMALP, open grey squares are CD81-DDM. Data are mean ± SEM, n = 3.

    Article Snippet: CD81 protein samples were bound to plates, blocked and washed as described above.

    Techniques: Purification, Spectroscopy, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay

    CD81 can be solubilised using SMA polymers or conventional detergent. Membrane preparations from CD81-expressing P. pastoris cells (30 mg/ml wet pellet weight) were solubilised with 2% (w/v) DDM, 2.5% (w/v) SMA polymer or PBS (negative control) for 1 h at room temperature. A) Image showing the change in appearance as SMA clarifies the membrane suspension compared to PBS control. B) Optical density measurements of the samples measured at 600 nm. Data are mean ± SD, n = 3. C) Following solubilisation, samples were centrifuged at 100,000 g for 20 min at 4 °C. Solubilised protein in the supernatant (S) was harvested. Insoluble material in the pellet (P) was resuspended in an equal volume of purification buffer supplemented with 2% (w/v) SDS. Samples were analysed by Western blot using an anti-CD81 primary antibody (mAb 2s131). D) Average CD81 solubilisation efficiency. Data are mean ± SD, n = 3.

    Journal: Biochimica et Biophysica Acta. Biomembranes

    Article Title: CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups

    doi: 10.1016/j.bbamem.2020.183419

    Figure Lengend Snippet: CD81 can be solubilised using SMA polymers or conventional detergent. Membrane preparations from CD81-expressing P. pastoris cells (30 mg/ml wet pellet weight) were solubilised with 2% (w/v) DDM, 2.5% (w/v) SMA polymer or PBS (negative control) for 1 h at room temperature. A) Image showing the change in appearance as SMA clarifies the membrane suspension compared to PBS control. B) Optical density measurements of the samples measured at 600 nm. Data are mean ± SD, n = 3. C) Following solubilisation, samples were centrifuged at 100,000 g for 20 min at 4 °C. Solubilised protein in the supernatant (S) was harvested. Insoluble material in the pellet (P) was resuspended in an equal volume of purification buffer supplemented with 2% (w/v) SDS. Samples were analysed by Western blot using an anti-CD81 primary antibody (mAb 2s131). D) Average CD81 solubilisation efficiency. Data are mean ± SD, n = 3.

    Article Snippet: CD81 protein samples were bound to plates, blocked and washed as described above.

    Techniques: Expressing, Negative Control, Purification, Western Blot

    Purification of SMALP-encapsulated CD81 yields a functionally-folded protein. A) The Ni-NTA affinity purification procedure was modified to increase the amount of resin used, and increase the volume of washes. Samples of each step of the purification were analysed by SDS-PAGE stained with Instant Blue. Sol is the total solubilised protein, Pt is the insoluble protein, FT is flow-through and numbers show the concentration in mM of imidazole in the wash buffers. B) Elution fractions containing purified CD81 were pooled together and their folded state assessed by an ELISA using conformation-sensitive anti-CD81 antibodies; 1s337 and 1s135, alongside the linear peptide antibody 2s131. Bovine serum albumin was used as a negative control. Primary antibodies were detected with an anti-mouse-HRP secondary antibody and visualised by SIGMA FAST ™ OPD tablet. Optical density at 492 nm was measured. Data are mean ± SD, n = 2. Purified CD81 was also analysed for binding to the E2 glycoprotein (that acts as a CD81 ligand) by ELISA , n = 3.

    Journal: Biochimica et Biophysica Acta. Biomembranes

    Article Title: CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups

    doi: 10.1016/j.bbamem.2020.183419

    Figure Lengend Snippet: Purification of SMALP-encapsulated CD81 yields a functionally-folded protein. A) The Ni-NTA affinity purification procedure was modified to increase the amount of resin used, and increase the volume of washes. Samples of each step of the purification were analysed by SDS-PAGE stained with Instant Blue. Sol is the total solubilised protein, Pt is the insoluble protein, FT is flow-through and numbers show the concentration in mM of imidazole in the wash buffers. B) Elution fractions containing purified CD81 were pooled together and their folded state assessed by an ELISA using conformation-sensitive anti-CD81 antibodies; 1s337 and 1s135, alongside the linear peptide antibody 2s131. Bovine serum albumin was used as a negative control. Primary antibodies were detected with an anti-mouse-HRP secondary antibody and visualised by SIGMA FAST ™ OPD tablet. Optical density at 492 nm was measured. Data are mean ± SD, n = 2. Purified CD81 was also analysed for binding to the E2 glycoprotein (that acts as a CD81 ligand) by ELISA , n = 3.

    Article Snippet: CD81 protein samples were bound to plates, blocked and washed as described above.

    Techniques: Purification, Affinity Purification, Modification, SDS Page, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Binding Assay

    Solubilisation of CD81 by SMA 2000 is slower than the break-up of the total membrane. A) Representative Western blot showing the amount of CD81 remaining insoluble (in the pellet) after various lengths of incubation with 2.5% (w/v) SMA 2000 at room temperature. B) Average measurements of solubilisation at different time points. Optical density measurements at 600 nm (solid black circles). Percentage insoluble CD81 determined by densitometric analysis of Western blots as shown in A (open grey triangles). Data are mean ± SD, n = 3. C) Representative Western blot showing the amount of CD81 remaining insoluble after incubation with varying concentrations of SMA 2000 for 1 h at room temperature. D) Average measurements of solubilisation using different SMA 2000 concentrations. Optical density measurements at 600 nm (solid black circles). Percentage insoluble CD81 determined by densitometric analysis of Western blots as shown in C (open grey triangles). Data are mean ± SD, n = 2.

    Journal: Biochimica et Biophysica Acta. Biomembranes

    Article Title: CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups

    doi: 10.1016/j.bbamem.2020.183419

    Figure Lengend Snippet: Solubilisation of CD81 by SMA 2000 is slower than the break-up of the total membrane. A) Representative Western blot showing the amount of CD81 remaining insoluble (in the pellet) after various lengths of incubation with 2.5% (w/v) SMA 2000 at room temperature. B) Average measurements of solubilisation at different time points. Optical density measurements at 600 nm (solid black circles). Percentage insoluble CD81 determined by densitometric analysis of Western blots as shown in A (open grey triangles). Data are mean ± SD, n = 3. C) Representative Western blot showing the amount of CD81 remaining insoluble after incubation with varying concentrations of SMA 2000 for 1 h at room temperature. D) Average measurements of solubilisation using different SMA 2000 concentrations. Optical density measurements at 600 nm (solid black circles). Percentage insoluble CD81 determined by densitometric analysis of Western blots as shown in C (open grey triangles). Data are mean ± SD, n = 2.

    Article Snippet: CD81 protein samples were bound to plates, blocked and washed as described above.

    Techniques: Western Blot, Incubation

    Biophysical characterisation of purified CD81. A) DLS analysis of purified CD81 either encapsulated within SMALPs (black) or within DDM micelles (grey) obtained using a Malvern Instruments Zetasizer Nano S (633 nm) with a 1.0 cm path length disposable cuvette. Seven technical replicates were performed for each sample, n = 2. B) CD spectra of purified CD81 either encapsulated within SMALPs (black) or within DDM micelles (grey). Purified CD81-SMALP and CD81-DDM was buffer exchanged into 20 mM sodium phosphate buffer pH 8 at concentrations of 0.05 mg/ml and 0.09 mg/ml, respectively. 200 μl of each sample was used in a 1 mm path length cuvette and measured using a Jasco J-1500 instrument. Maximum absorbance of 260 nm and minimum absorbance of 180 nm was used for CD detection at 0.2 nm intervals at 20 °C; 18 technical replicates were performed for each sample, where n = 2.

    Journal: Biochimica et Biophysica Acta. Biomembranes

    Article Title: CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups

    doi: 10.1016/j.bbamem.2020.183419

    Figure Lengend Snippet: Biophysical characterisation of purified CD81. A) DLS analysis of purified CD81 either encapsulated within SMALPs (black) or within DDM micelles (grey) obtained using a Malvern Instruments Zetasizer Nano S (633 nm) with a 1.0 cm path length disposable cuvette. Seven technical replicates were performed for each sample, n = 2. B) CD spectra of purified CD81 either encapsulated within SMALPs (black) or within DDM micelles (grey). Purified CD81-SMALP and CD81-DDM was buffer exchanged into 20 mM sodium phosphate buffer pH 8 at concentrations of 0.05 mg/ml and 0.09 mg/ml, respectively. 200 μl of each sample was used in a 1 mm path length cuvette and measured using a Jasco J-1500 instrument. Maximum absorbance of 260 nm and minimum absorbance of 180 nm was used for CD detection at 0.2 nm intervals at 20 °C; 18 technical replicates were performed for each sample, where n = 2.

    Article Snippet: CD81 protein samples were bound to plates, blocked and washed as described above.

    Techniques: Purification

    Positive ion mode MS spectra measured for total lipid extracts of total yeast membrane, SMA solubilised membrane and purified CD81 SMALP. Identified glycerophosphatidylcholines are labelled in green (PC), glycerophosphatidyletanolamines in blue (PE), glycerophosphatidylinositols in magenta (PI), glycerophosphatidycacids in peach (PA) and sphingomyelin in orange (SM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Journal: Biochimica et Biophysica Acta. Biomembranes

    Article Title: CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups

    doi: 10.1016/j.bbamem.2020.183419

    Figure Lengend Snippet: Positive ion mode MS spectra measured for total lipid extracts of total yeast membrane, SMA solubilised membrane and purified CD81 SMALP. Identified glycerophosphatidylcholines are labelled in green (PC), glycerophosphatidyletanolamines in blue (PE), glycerophosphatidylinositols in magenta (PI), glycerophosphatidycacids in peach (PA) and sphingomyelin in orange (SM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: CD81 protein samples were bound to plates, blocked and washed as described above.

    Techniques: Purification

    Biophysical characterisation of CD81–SMALP following size exclusion chromatography. A) CD spectra of affinity purified CD81-SMALP prior to SEC (black), CD81-SMALP in peak 1 following SEC (light grey) and from peak 2 following SEC (dark grey, dashed). 200 μl of each sample was used in a 1 mm path length cuvette and measured using a Jasco J-1500 instrument. Maximum absorbance of 260 nm and minimum absorbance of 180 nm was used for CD detection at 0.2 nm intervals at 20 °C; 18 technical replicates were performed for each sample, where n = 2. B) DLS analysis of purified CD81-SMALP prior to SEC (black), CD81-SMALP in peak 1 following SEC (light grey) and from peak 2 following SEC (dark grey, dashed). Data were obtained using a Malvern Instruments Zetasizer Nano S (633 nm) with 1.0 cm path length disposable cuvette. Seven technical replicates were performed for each sample, n = 2.

    Journal: Biochimica et Biophysica Acta. Biomembranes

    Article Title: CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups

    doi: 10.1016/j.bbamem.2020.183419

    Figure Lengend Snippet: Biophysical characterisation of CD81–SMALP following size exclusion chromatography. A) CD spectra of affinity purified CD81-SMALP prior to SEC (black), CD81-SMALP in peak 1 following SEC (light grey) and from peak 2 following SEC (dark grey, dashed). 200 μl of each sample was used in a 1 mm path length cuvette and measured using a Jasco J-1500 instrument. Maximum absorbance of 260 nm and minimum absorbance of 180 nm was used for CD detection at 0.2 nm intervals at 20 °C; 18 technical replicates were performed for each sample, where n = 2. B) DLS analysis of purified CD81-SMALP prior to SEC (black), CD81-SMALP in peak 1 following SEC (light grey) and from peak 2 following SEC (dark grey, dashed). Data were obtained using a Malvern Instruments Zetasizer Nano S (633 nm) with 1.0 cm path length disposable cuvette. Seven technical replicates were performed for each sample, n = 2.

    Article Snippet: CD81 protein samples were bound to plates, blocked and washed as described above.

    Techniques: Size-exclusion Chromatography, Affinity Purification, Purification

    Characterization of exosomes isolated from A549 cells (Exo-A549) or ZIKV infected A549 cells (Exo-ZIKV). Transmission electron micrograph of Exo-A549 (A) and Exo-ZIKV (B). Nanoparticle tracking analysis of exosomes indicated Exo-A549 particles sizes median diameter of 125.3 nm (C) and Exo-ZIKV median size diameter of 127.2 nm (D). Peak analysis of exosomes (E). Western blotting of exosomes was performed to confirm the presence of exosomal marker protein, CD63, CD81 and TSG101. Absence the endoplasmic reticulum protein, Calnexin, in exosomes but was detectable in whole cell lysates (F).

    Journal: Life Sciences

    Article Title: DEFA1B inhibits ZIKV replication and retards cell cycle progression through interaction with ORC1

    doi: 10.1016/j.lfs.2020.118564

    Figure Lengend Snippet: Characterization of exosomes isolated from A549 cells (Exo-A549) or ZIKV infected A549 cells (Exo-ZIKV). Transmission electron micrograph of Exo-A549 (A) and Exo-ZIKV (B). Nanoparticle tracking analysis of exosomes indicated Exo-A549 particles sizes median diameter of 125.3 nm (C) and Exo-ZIKV median size diameter of 127.2 nm (D). Peak analysis of exosomes (E). Western blotting of exosomes was performed to confirm the presence of exosomal marker protein, CD63, CD81 and TSG101. Absence the endoplasmic reticulum protein, Calnexin, in exosomes but was detectable in whole cell lysates (F).

    Article Snippet: Primary antibodies of CD63, CD81, TSG101, DEFA1B and ORC1 were obtained from Sigma-Aldrich (St Louis, MO, USA), and antibodies of Calnexin, NS1, PARP and GAPDH were obtain from CST (MA, USA), DEFA1B antibody was from proteintech (IL, USA).

    Techniques: Isolation, Infection, Transmission Assay, Western Blot, Marker

    DENV-induced B cell activation depends on CD81 activation. B lymphocytes were mock-treated or cultured with DENV2 (MOI = 1) in the presence or absence of anti-CD81 neutralizing antibody. A) At 12 days post infection, the supernatants were harvested and IgM levels were measured by ELISA. Data are representative of four independent experiments. Statistical analysis were performed and p values are indicated in the figures. B) The cells were harvested after different time points and DENV RNA levels were evaluated by qRT-PCR. Data are representative of three independent experiments. C) After 48h, the cells were harvested and the expression of ERK, p38 and JNK MAPK, phosphorylated (phospho) or not (unphospho) were analyzed in the cell lysates by western blotting, as indicated. The bars indicate the ratio between the analyzed phosphorylated protein and the corresponding unphosphorylated one; dots represent individual data.

    Journal: PLoS ONE

    Article Title: Dengue Virus Directly Stimulates Polyclonal B Cell Activation

    doi: 10.1371/journal.pone.0143391

    Figure Lengend Snippet: DENV-induced B cell activation depends on CD81 activation. B lymphocytes were mock-treated or cultured with DENV2 (MOI = 1) in the presence or absence of anti-CD81 neutralizing antibody. A) At 12 days post infection, the supernatants were harvested and IgM levels were measured by ELISA. Data are representative of four independent experiments. Statistical analysis were performed and p values are indicated in the figures. B) The cells were harvested after different time points and DENV RNA levels were evaluated by qRT-PCR. Data are representative of three independent experiments. C) After 48h, the cells were harvested and the expression of ERK, p38 and JNK MAPK, phosphorylated (phospho) or not (unphospho) were analyzed in the cell lysates by western blotting, as indicated. The bars indicate the ratio between the analyzed phosphorylated protein and the corresponding unphosphorylated one; dots represent individual data.

    Article Snippet: To further confirm the data, the viability of B cells cultured with anti-CD81 were also assessed by XTT assay (Sigma Aldrich), according to the manufacturer’s protocol; and the concentration of lactate dehydrogenase (LDH) in culture supernatant were evaluated, using LDH assay Kit (Doles, Goias, Brasil).

    Techniques: Activation Assay, Cell Culture, Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Western Blot

    Caveolin-1 promotes Ag-induced membrane raft synaptic polarization and actin polymerization in CD8 + T cells. A, Left panel , Phase and fluorescent microscopy of raft-associated GM1 glycolipid localization in CD8 + cells from OT-1 cav-1 +/+ and cav-1 −/−

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Caveolin-1 Orchestrates TCR Synaptic Polarity, Signal Specificity, and Function in CD8 T Cells

    doi: 10.4049/jimmunol.1101447

    Figure Lengend Snippet: Caveolin-1 promotes Ag-induced membrane raft synaptic polarization and actin polymerization in CD8 + T cells. A, Left panel , Phase and fluorescent microscopy of raft-associated GM1 glycolipid localization in CD8 + cells from OT-1 cav-1 +/+ and cav-1 −/−

    Article Snippet: Purified wild-type or cav-1−/− CD8 cells were expanded on APCs for 3 d. Cells were then transduced with caveolin-1–expressing viral supernatant by spinning cells for 90 min in the presence of 8 µg/ml polybrene (Millipore).

    Techniques: Microscopy

    Only caveolin-1–deficient CD8 + T cells are defective at TCR/CD28-mediated proliferation and IFN-γ and CTL effector function. A , cav-1 +/+ (black bar) or cav-1 −/− (white bar) CD8 cells stimulated for 72 h with CD3/CD28-specific

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Caveolin-1 Orchestrates TCR Synaptic Polarity, Signal Specificity, and Function in CD8 T Cells

    doi: 10.4049/jimmunol.1101447

    Figure Lengend Snippet: Only caveolin-1–deficient CD8 + T cells are defective at TCR/CD28-mediated proliferation and IFN-γ and CTL effector function. A , cav-1 +/+ (black bar) or cav-1 −/− (white bar) CD8 cells stimulated for 72 h with CD3/CD28-specific

    Article Snippet: Purified wild-type or cav-1−/− CD8 cells were expanded on APCs for 3 d. Cells were then transduced with caveolin-1–expressing viral supernatant by spinning cells for 90 min in the presence of 8 µg/ml polybrene (Millipore).

    Techniques: CTL Assay

    Caveolin-1 knockout mice are deficient at Ag-specific CD8 + T cell expansion in response to LCMV infection. A , Flow cytometry profiles (CD8 versus GP 33–41 /D b tetramer) of CD8 + gated splenocytes from day 7 post–LCMV-infected cav-1 +/+ ( left

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Caveolin-1 Orchestrates TCR Synaptic Polarity, Signal Specificity, and Function in CD8 T Cells

    doi: 10.4049/jimmunol.1101447

    Figure Lengend Snippet: Caveolin-1 knockout mice are deficient at Ag-specific CD8 + T cell expansion in response to LCMV infection. A , Flow cytometry profiles (CD8 versus GP 33–41 /D b tetramer) of CD8 + gated splenocytes from day 7 post–LCMV-infected cav-1 +/+ ( left

    Article Snippet: Purified wild-type or cav-1−/− CD8 cells were expanded on APCs for 3 d. Cells were then transduced with caveolin-1–expressing viral supernatant by spinning cells for 90 min in the presence of 8 µg/ml polybrene (Millipore).

    Techniques: Knock-Out, Mouse Assay, Infection, Flow Cytometry, Cytometry

    Caveolin-1 selectively modulates TCR-induced transcriptional activation in CD8 + T cells. A , Purified cav-1 +/+ (solid line) or cav-1 −/− (dashed line) CD8 + T cells were stimulated with Abs to CD3/CD28 and harvested at the various time points

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Caveolin-1 Orchestrates TCR Synaptic Polarity, Signal Specificity, and Function in CD8 T Cells

    doi: 10.4049/jimmunol.1101447

    Figure Lengend Snippet: Caveolin-1 selectively modulates TCR-induced transcriptional activation in CD8 + T cells. A , Purified cav-1 +/+ (solid line) or cav-1 −/− (dashed line) CD8 + T cells were stimulated with Abs to CD3/CD28 and harvested at the various time points

    Article Snippet: Purified wild-type or cav-1−/− CD8 cells were expanded on APCs for 3 d. Cells were then transduced with caveolin-1–expressing viral supernatant by spinning cells for 90 min in the presence of 8 µg/ml polybrene (Millipore).

    Techniques: Activation Assay, Purification

    Caveolin-1 expression is required for generation of optimal virus-specific CD8 + T cell function and viral clearance. Cav-1 +/+ and cav-1 −/− mice were injected with 2 × 10 5 PFU LCMV Armstrong strain i.p. A , Flow profiles (CD8 versus

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Caveolin-1 Orchestrates TCR Synaptic Polarity, Signal Specificity, and Function in CD8 T Cells

    doi: 10.4049/jimmunol.1101447

    Figure Lengend Snippet: Caveolin-1 expression is required for generation of optimal virus-specific CD8 + T cell function and viral clearance. Cav-1 +/+ and cav-1 −/− mice were injected with 2 × 10 5 PFU LCMV Armstrong strain i.p. A , Flow profiles (CD8 versus

    Article Snippet: Purified wild-type or cav-1−/− CD8 cells were expanded on APCs for 3 d. Cells were then transduced with caveolin-1–expressing viral supernatant by spinning cells for 90 min in the presence of 8 µg/ml polybrene (Millipore).

    Techniques: Expressing, Cell Function Assay, Mouse Assay, Injection, Flow Cytometry

    Caveolin-1 expression by Ag-specific CD8 + T cells is required for optimal CD8 + T cell expansion and effector function in vivo. Purified cav-1 +/+ or cav-1 −/− OT-1 CD8 + T cells were adoptively transferred into wild-type C57BL/6 mice that

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Caveolin-1 Orchestrates TCR Synaptic Polarity, Signal Specificity, and Function in CD8 T Cells

    doi: 10.4049/jimmunol.1101447

    Figure Lengend Snippet: Caveolin-1 expression by Ag-specific CD8 + T cells is required for optimal CD8 + T cell expansion and effector function in vivo. Purified cav-1 +/+ or cav-1 −/− OT-1 CD8 + T cells were adoptively transferred into wild-type C57BL/6 mice that

    Article Snippet: Purified wild-type or cav-1−/− CD8 cells were expanded on APCs for 3 d. Cells were then transduced with caveolin-1–expressing viral supernatant by spinning cells for 90 min in the presence of 8 µg/ml polybrene (Millipore).

    Techniques: Expressing, In Vivo, Purification, Mouse Assay