Journal: Microbiology Spectrum

Article Title: Density Analysis of Enterovirus D68 Shows Viral Particles Can Associate with Exosomes

doi: 10.1128/spectrum.02452-21

Figure Lengend Snippet: Immune recognition of various EV-D68 densities and characterization of membrane-associated virus. (A) The y axis represents average dilutions of anti-EV-D68 mouse serum required to neutralize virus, divided by the average TCID 50 for respective viral densities (ANOVA post hoc Student'’s t test, P = 0.42, P = 0.68, P = 0.70). (B) Three viral density isolates (1.11, 1.20, and 1.24 g/cm 3 ) were treated with 0.01 mg/mL 15C5-Chmra antibody for 1 h, then mix was put onto TCID 50 plates to assess the viral titer of each isolate. Gray highlight represents detection limit. Asterisks (*) indicate statistical significance (1.11 g/cm 3 , P = 0.0003; 1.20 g/cm 3 , P < 0.0001; 1.24 g/cm 3 , P = 0.0064), all versus respective control, determined by Dunnett’s Method. (C) 15C5-Chmra antibody bound to magnetic beads was added to membrane-associated and naked virus. After 1 h, a magnet was used to remove antibody and the supernatant was added to a TCID 50 plate to assess viral titer (15C5-Chmra versus control: *, P < 0.0005 for both membrane-associated and naked virus; Dunnett’s Method). (D) ICAM-5 or N -acetylneuraminic acid (sialic acid) were attached to magnetic beads and the antibody/bead complex was incubated with membrane-associated or naked virus samples for 1 h. Beads were rinsed twice in excess PBS and viral titer was assessed to determine how much virus was immunoprecipitated from the supernatant (control versus ICAM5 and control versus sialic acid for membrane-associated and naked virus; *, P = 0.0001 determined by Dunnett’s Method). (E) Exosome antibody array on 1.11 g/cm 3 fraction, examining cytosolic proteins (FLOT1, ALIX, TSG101), transmembrane proteins (CD63, CD81, ANXA5), and cis -golgi matrix protein as markers for cellular contamination (GM130). Example blot is shown on the right and chart represents average intensity across three biological replicates. Positive control indicates detection reagents are working correctly, and do not represent an exosome-specific control. Error bars represent standard deviation. Statistics: comparison with control (blank) using Dunnett’s Method ( P = 0.999 for GM130; *, P = 0.027 for FLOT1; P = 0.218 for ICAM; *, P = 0.005 for ALIX; P = 0.086 for CD81; *, P < 0.0001 for CD63; P = 0.305 for EpCAM; *, P < 0.0001 for ANXA5; *, P = 0.0008 for TSG101). Asterisks indicate statistical significance. (F) Anti-CD81 or anti-CD63 antibodies were attached to magnetic beads and incubated with membrane-associated virus. Supernatant was discarded, and beads were rinsed and treated with 0.01% NP-40 (to dissolve exosomes and release virus from bead) before TCID 50 measurement. CD81 versus control: *, P = 0.0178; CD63 versus control: *, P = 0.0180 as determined by Dunnett’s Method. Gray highlight represents detection limit. (G) RD or SH-SY5Y cells in TCID 50 plate were infected with MO47 with or without exosomes in the medium. The “A549 exosomes added” bar represents exosome-depleted media to which purified A549 exosomes were added. Gray panel represents TCID 50 plates containing SH-SY5Y cells. Each condition represents 3 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05. Green panel represents TCID 50 plates containing RD cells. Each condition represents 4 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05.

Article Snippet: We followed the coupling protocol from the Dynabeads Antibody Coupling Kit (Thermo Fisher, cat no. 14311D) to covalently attach magnetic beads to the following antibodies: anti-CD81 (1D6) monoclonal antibody (Novus Biologicals NB100-65805), anti-CD63 (H5C6) monoclonal antibody (Novus Biologicals NBP2-42225), 15C5-chimeric monoclonal antibody (generous gift from Michael Pauly at ZabBio), and anti-HSV negative control (generous gift from Michael Pauly at ZabBio).

Techniques: Membrane, Virus, Control, Magnetic Beads, Incubation, Immunoprecipitation, Ab Array, Positive Control, Standard Deviation, Comparison, Infection, Purification

Journal: PLoS ONE

Article Title: Antiviral Role of IFITM Proteins in African Swine Fever Virus Infection

doi: 10.1371/journal.pone.0154366

Figure Lengend Snippet: (A). Confocal microscopy images of Vero-IFITM cells or controls containing the empty vector stained with endosomal markers EEA1 (EE), CD63 (MVB), Rab7 (LE) and Lamp1 (LY). Endosomes were predominantly dispersed in the cytoplasm of cells containing the empty vector or concentrated to the perinuclear area in Vero-IFITM2 and 3 cells. (B). The change in distribution was quantified by measuring the mean distance to the nucleus of the different endosomal markers in x, y and z planes as described in materials and methods section. As shown in graphics, distance was reduced in Vero-IFITM2 and 3 cells. Graphics depict mean±SD of N = 30 cells per condition. Statistical significance was evaluated by a one-way ANOVA followed by Bonferroni’s multiple comparison test. Differences are marked with asterisks as indicated (* p <0.05; ** p <0.01).

Article Snippet: Escribano, INIA); ASFV mouse monoclonal antibodies anti-p72 (clone 1BC11 for immunofluorescence 1:1,000 or clone 18BG3 for WB 1:2,000) and anti-p150 (clone 17AH2, Ingenasa), 1:1,000; mouse monoclonal to CD63 (Developmental Studies Hybridoma Bank, University of Iowa, clone H5C6), 1:200; rabbit polyclonal to Lamp1 (Abcam), 1:50; rabbit polyclonal to EEA1 and Rab7 (Cell Signalling), 1:50.

Techniques: Confocal Microscopy, Plasmid Preparation, Staining, Comparison

Journal: PLoS ONE

Article Title: Antiviral Role of IFITM Proteins in African Swine Fever Virus Infection

doi: 10.1371/journal.pone.0154366

Figure Lengend Snippet: (A). Expression of IFITM1, 2 and 3 proteins (green) and CD63-positive late endosomes (red) in Vero-IFITM cells or cells containing the empty vector. (B). Distribution of IFITM2 (green) and CD63 (red) in cells containing the empty vector. Bar = 10μm. (C). Data were plotted on graphics representing the colocalization values between IFITM2 and endosomes in Vero-IFITM2 relative to control cells of N = 30 cells per condition. Statistical significance was evaluated by a one-way ANOVA followed by Bonferroni’s multiple comparison test. Differences are marked with asterisks as indicated (*** p <0.001).

Article Snippet: Escribano, INIA); ASFV mouse monoclonal antibodies anti-p72 (clone 1BC11 for immunofluorescence 1:1,000 or clone 18BG3 for WB 1:2,000) and anti-p150 (clone 17AH2, Ingenasa), 1:1,000; mouse monoclonal to CD63 (Developmental Studies Hybridoma Bank, University of Iowa, clone H5C6), 1:200; rabbit polyclonal to Lamp1 (Abcam), 1:50; rabbit polyclonal to EEA1 and Rab7 (Cell Signalling), 1:50.

Techniques: Expressing, Plasmid Preparation, Control, Comparison

Journal: The Journal of Cell Biology

Article Title: Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

doi: 10.1083/jcb.201703206

Figure Lengend Snippet: CD63-pHluorin is sorted into acidic MVBs and released via exosomes. (a) Proposed model for the visualization of MVB–PM fusion: a pH-sensitive optical reporter (CD63-pHluorin) is quenched when facing the acidic lumen of the MVB. Upon fusion, low luminal pH is immediately neutralized, resulting in a sudden increase in fluorescent intensity. EC, extracellular. (b) Immunofluorescent colabeling of total CD63 (red) and CD63-pHluorin (green) in HeLa cells. PCC, Pearson’s correlation coefficient. (c) TIRF images of a CD63-pHluorin–expressing HeLa cell at normal and elevated intracellular pH (NH 4 Cl superfusion). On the right, a heat map revealing acidic vesicles close to the PM was obtained by subtracting the fluorescent intensity values of the normal pH from the high-pH condition. (d) EM images of an MVB close to the PM (left) and EVs aligning the PM (right) labeled with gold particles directed to GFP (10 nm) in CD63-pHluorin–expressing HeLa cells. (e) Imaging flow cytometry of the number of late endosomes per cell in a 2.5-µm optical section in CD63-pHluorin–expressing cells (left) or immunostaining against LAMP1 in nontransfected cells (right; n > 2,000 cells). (f) Volume distribution of endosomes based on analysis of whole-cell confocal scans (error bars represent SD; n = 3). The blue area accounts for 75% of the total number of endosomes and covers the 400–600-nm-diameter range. (g) Immunogold labeling on purified exosomes with gold particles (10 nm) coupled to anti-GFP antibody. (h) Western blotting analysis on untransfected (−) and CD63-pHluorin–transfected (+) cells and purified exosomes for total CD63 and GFP. (i) Example of a localized sudden increase in fluorescence at the PM before the event (1), during the event (2), and right before disappearance of the signal (3). (j) Left: total projection of fusion events (bright spots) over a time course of 3 min onto two cells (blue). Right: representative example of CD63-pHluorin–expressing HeLa cell. N, nucleus. Bars: (b, c, and j) 10 µm; (i) 2.5 µm. (k) Effect of incubation with GW4896 (5 µM; n ≥ 8 cells per condition) and nSMase-2 knockdown ( n ≥ 22 cells per condition) on fusion activity in HeLa cells. *, P < 0.05; ***, P < 0.001 using Student’s two-tailed two-sample t test. Whiskers in the box plots represent 1.5 times the interquartile distance or the highest or lowest point, whichever is shorter. (l) Western blotting analysis on purified exosomes from GW4896- and control-treated HeLa cells for CD63 and CD81.

Article Snippet: Monoclonal antibodies against CD63 (mouse; H5C6; 556019; BD), CD81 (mouse; JS-81; 551112; BD), CD9 (mouse; a gift from E. Rubinstein, Institut National de la Santé et de la Recherché Médicale, Villejuif, France), and Alix (3A9; 2171; Cell Signaling Technology) were used at 1:200.

Techniques: Expressing, Labeling, Imaging, Flow Cytometry, Immunostaining, Purification, Western Blot, Transfection, Fluorescence, Incubation, Activity Assay, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

doi: 10.1083/jcb.201703206

Figure Lengend Snippet: CD63-pHluorin fusion events are derived from MVBs. (a) Left three panels: live imaging of fusion events (indicated by white arrows) over a time course of 12 s onto one cell before the event (left), at the start of the event (middle), and right before fixation of the cell (3). Right: inset showing a magnification of the localized sudden increase in fluorescence at the PM (highlighted by a dashed line square) right before fixation. (b) Left: correlation of light microscopy signal of a fusion event observed by live imaging with EM pictures of the first section of the cell facing the coverslip (low magnification). Right: correlation of light microscopy signal with the first slice of the electron tomographic reconstruction of the first section of the cell facing the coverslip. The orange circle indicates the error range (167 nm) of the correlation performed by eC-CLEM. (c) 3D model of the electron tomographic reconstruction. The ER is depicted in light violet. Dense compartments are depicted in brown. The structure of interest is depicted in red and orange. (d) Bottom side view of the 3D model of the compartment of interest in its surroundings. The white arrow indicates the opening of the MVB where ILVs are released. (e) 3D model showing the MVB isolated from its environment. ILVs secreted through the opening of the MVB are depicted in white. (f) Top view of the secretory profile of the MVB that correlates with the fluorescence burst of the CD63-pHluorin fusion event.

Article Snippet: Monoclonal antibodies against CD63 (mouse; H5C6; 556019; BD), CD81 (mouse; JS-81; 551112; BD), CD9 (mouse; a gift from E. Rubinstein, Institut National de la Santé et de la Recherché Médicale, Villejuif, France), and Alix (3A9; 2171; Cell Signaling Technology) were used at 1:200.

Techniques: Derivative Assay, Imaging, Fluorescence, Light Microscopy, Isolation

Journal: The Journal of Cell Biology

Article Title: Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

doi: 10.1083/jcb.201703206

Figure Lengend Snippet: MVB–PM fusion is distinct from other forms of vesicle-mediated exocytosis. (a) Schematic model showing the markers used in this study for the different types of cargo delivery of vesicles fusing with the PM. (b) Time-lapse imaging (heat maps) of a fusion event of the exosomal protein CD63-pHluorin. (c) Time-lapse images of soluble (NPY-pHluorin) and membrane protein (VAMP2-pHluorin) fusion events. (d) Fluorescent signal duration of NPY (mean = 0.85 s), VAMP2 (mean = 2.12 s), and CD63 (mean = 106.55 s) fusion events. n ≥ 13 events per reporter. (e) 3D heat maps of three consecutive CD63-pHluorin fusion event frames. (f) Western blot for exosomal markers (CD63 and Alix) on EVs purified from the supernatant (soluble) and EVs attached to the cell surface (PM attached) isolated after short trypsinization of the cells. (g) Direct comparison between signal duration of fusion events of CD81- and CD9-pHluorin relative to CD63-pHluorin. n ≥ 20 events per reporter. ***, P < 0.001; ****, P < 0.0001 using Student’s two-tailed two-sample t test.

Article Snippet: Monoclonal antibodies against CD63 (mouse; H5C6; 556019; BD), CD81 (mouse; JS-81; 551112; BD), CD9 (mouse; a gift from E. Rubinstein, Institut National de la Santé et de la Recherché Médicale, Villejuif, France), and Alix (3A9; 2171; Cell Signaling Technology) were used at 1:200.

Techniques: Imaging, Western Blot, Purification, Isolation, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

doi: 10.1083/jcb.201703206

Figure Lengend Snippet: GPCR activation triggers MVB–PM fusion in single cells in a calcium-independent manner. (a) Schematic model of imaging setup. (b) Fusion activity of HeLa cells stimulated with KCl (70 mM), caffeine (20 mM), or histamine (100 µM). n ≥ 8 cells per condition. (c) Total projection of fusion events over a 60-s time course onto cells before (top) and after (bottom) stimulation with histamine (100 µM). Pseudocolored as in . (d) Measurement of individual HeLa cells ( n = 14) before and during stimulation with histamine (100 µM). (e) Mean fusion kinetics of CD63-pHluorin HeLa cells ( n = 6) showing the distribution of fusion events over time (dark blue line; SD is in light blue) and the calcium levels (red) during histamine stimulation (gray-shaded block). (f) Heat maps revealing calcium responses (measured by Fluo-4) upon histamine stimulation obtained by subtracting the fluorescent intensity values before stimulation from those after 8-s stimulation. Cells were nontreated or incubated with a buffer with fast (BAPTA) or slow (EGTA) calcium-binding kinetics. Bars, 10 µm. (g) Quantification of fusion activity of histamine-stimulated HeLa cells in the presence of EGTA (top) or BAPTA (bottom) buffers. n ≥ 10 cells per condition. (h) Measurement of individual HUVEC cells ( n = 30) before and after stimulation with histamine (100 µM). *, P < 0.05; **, P < 0.01 using Student’s two-tailed two-sample t test. All t tests were unpaired except for d and h. Whiskers in the box plots (b and g) represent 1.5 times the interquartile distance or the highest or lowest point, whichever is shorter.

Article Snippet: Monoclonal antibodies against CD63 (mouse; H5C6; 556019; BD), CD81 (mouse; JS-81; 551112; BD), CD9 (mouse; a gift from E. Rubinstein, Institut National de la Santé et de la Recherché Médicale, Villejuif, France), and Alix (3A9; 2171; Cell Signaling Technology) were used at 1:200.

Techniques: Activation Assay, Imaging, Activity Assay, Blocking Assay, Incubation, Binding Assay, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

doi: 10.1083/jcb.201703206

Figure Lengend Snippet: The GPCR downstream effector SNAP23 regulates MVB–PM fusion. (a) Network of proteins of interest together with direct interactors with altered phosphorylation levels upon histamine (100 µM) stimulation as identified by phosphoproteomics in HeLa and HUVEC cells. Proteins of interest are depicted with a blue rim. FC, fold change. (b) Graph showing the signal intensity values of phosphorylated peptides from proteins of interest before and after stimulation with 100 µM histamine. Data represent means ± SD of two technical replicates per condition. *, P < 0.05 (P = 0.048) using Student’s two-tailed two-sample t test. (c) Western blotting analysis on SNAP23 protein expression in six different cell lines. (d) Confocal analysis of FL (GFP-SNAP23-FL) and truncated (GFP-SNAP23-CΔ9) GFP-SNAP23 (in gray)–transfected SiHa cells labeled for CD63 (red). (e) Total projection of fusion events in CD63-pHluorin SiHa cells cotransfected with SNAP23-FL or SNAP23-CΔ9 over 3 min. Pseudocolored as in . Bars, 10 µm. (f) Quantification of fusion events in CD63-pHluorin SiHa cells cotransfected with SNAP23-FL or SNAP23-CΔ9. n ≥ 10 cells per condition. (g) Confirmation of SNAP23 knockdown (KD) at the protein level in HeLa cells. (h) Effect of SNAP23 knockdown on MVB–PM fusion in HeLa cells. n ≥ 17 cells per condition. (i) Confirmation of SNAP23 and syntaxin-4 knockdown in HeLa cells at the mRNA level. Data represent means ± SD. (j) Effect of the knockdown of SNAP23 or syntaxin-4 on the fusion activity of HeLa cells. n ≥ 11 cells per condition. ctrl, nontransfected; siCTRL, control siRNA. *, P < 0.05; **, P < 0.01 using Student’s two-tailed two-sample t test. Whiskers in the box plots (f, h, and j) represent 1.5 times the interquartile distance or the highest or lowest point, whichever is shorter.

Article Snippet: Monoclonal antibodies against CD63 (mouse; H5C6; 556019; BD), CD81 (mouse; JS-81; 551112; BD), CD9 (mouse; a gift from E. Rubinstein, Institut National de la Santé et de la Recherché Médicale, Villejuif, France), and Alix (3A9; 2171; Cell Signaling Technology) were used at 1:200.

Techniques: Two Tailed Test, Western Blot, Expressing, Transfection, Labeling, Activity Assay

Journal: The Journal of Cell Biology

Article Title: Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling

doi: 10.1083/jcb.201703206

Figure Lengend Snippet: GPCR activation triggers MVB–PM fusion in HeLa cells via SNAP23-Ser110 phosphorylation. (a) Fusion activity of histamine-stimulated cells nontreated or treated with the Gα q inhibitor UBO-QIC (1 µM). n ≥ 24 cells per condition. (b) Basal fusion activity in cells treated with PKC inhibitors GÖ6976 (1 µM) or GÖ6983 (1 µM). n ≥ 11 cells per condition. (c) Fusion activity of histamine-stimulated cells nontreated or preincubated with GÖ6983 (1 µM). n ≥ 11 cells per condition. (d) Schematic representation of SNAP23 with SNARE motifs, a membrane-anchoring domain (M), and all phosphosites with the posphosite targeted by histamine stimulation (Ser110) in bold. (e) Fusion activity of histamine-stimulated cells transfected with WT SNAP23, phosphomutant SNAP23-S110A, or phosphomimic SNAP23-S110D. n ≥ 16 cells per condition. (f) Left: fusion activity of CD63-, CD81-, and CD9-pHluorin HeLa cells cotransfected with SNAP23 WT or SNAP23-S110A. n ≥ 16 cells per condition. Western blot on exosomes isolated from SNAP23-WT and SNAP23-S110A HeLa cells labeled for CD63, CD9, CD81, flotillin-1, and syntenin-1. (g) Schematic representation of the histamine-stimulated pathway leading to exosome release as identified by phosphoproteomics and specific inhibitors. Blue-rimmed proteins represent the putative pathway implicated by both experiments. The IP3–Ca 2+ pathway is represented in gray as a direct link with MVB–PM fusion is missing. **, P < 0.01; ****, P < 0.0001 using Student’s two-tailed two-sample t test. All t tests were paired except for b and f. Whiskers in the box plots in b and f represent 1.5 times the interquartile distance or the highest or lowest point, whichever is shorter.

Article Snippet: Monoclonal antibodies against CD63 (mouse; H5C6; 556019; BD), CD81 (mouse; JS-81; 551112; BD), CD9 (mouse; a gift from E. Rubinstein, Institut National de la Santé et de la Recherché Médicale, Villejuif, France), and Alix (3A9; 2171; Cell Signaling Technology) were used at 1:200.

Techniques: Activation Assay, Activity Assay, Transfection, Western Blot, Isolation, Labeling, Two Tailed Test

Journal: Biology of reproduction

Article Title: Human villous trophoblasts express and secrete placenta-specific microRNAs into maternal circulation via exosomes.

doi: 10.1095/biolreprod.108.075481

Figure Lengend Snippet: FIG. 6. In vivo localization of MIR517B and CD63 in the human placenta. The same section of the full-term human placenta was subjected to ISH for MIR517B (A [red]) and subsequent immunostaining for CD63 (B [green]), a marker of the MVB/exosome. C) The merged image of MIR517B and CD63 signals with DAPI-stained nuclei (blue). Some MIR517B signals are colocalized with CD63-positive compartments (arrows) in the STB. The boundaries of STB and fetal capillaries (#) are indicated by white lines. Intervillous space (*) and villous stroma (VS) are indicated. Bar ¼ 10 lm.

Article Snippet: Some hybridized sections were subsequently immunostained with mouse anti-CD63 monoclonal antibodies (catalog number sc-5275, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA; and catalog number H5C6, 1:400; Developmental Studies Hybridoma Bank, Iowa City, IA).

Techniques: In Vivo, Immunostaining, Marker, Staining

Journal: Biology of reproduction

Article Title: Human villous trophoblasts express and secrete placenta-specific microRNAs into maternal circulation via exosomes.

doi: 10.1095/biolreprod.108.075481

Figure Lengend Snippet: FIG. 7. In vitro localization of MIRLET7B and CD63 in trophoblast cells. (A–C) Endogenous CD63 in BeWo cells. A) Immunostaining of CD63. The cells contain CD63-positive vesicular structures (arrows). B) DAPI-stained nuclei of the same cells. C) The merged image of CD63 and DAPI. D–F) Alexa Fluor 594-labeled MIRLET7B was introduced in BeWo cells together with the expression plasmid of EGFP-CD63 as a marker of the MVB/exosome. D) EFGP- CD63-transfected BeWo cells (green). E) Exogenously introduced MIRLET7B in the same EGFP-CD63-expressing cells (red). F) The merged image of CD63 and MIRLET7B signals. Some MIRLET7B signals are colocalized with CD63-positive compartments (arrows). MIRLET7B-negative CD63 compartments are also indicated (arrowheads). Bar ¼ 10 lm.

Article Snippet: Some hybridized sections were subsequently immunostained with mouse anti-CD63 monoclonal antibodies (catalog number sc-5275, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA; and catalog number H5C6, 1:400; Developmental Studies Hybridoma Bank, Iowa City, IA).

Techniques: In Vitro, Immunostaining, Staining, Labeling, Expressing, Plasmid Preparation, Marker, Transfection

Journal: Biology of reproduction

Article Title: Human villous trophoblasts express and secrete placenta-specific microRNAs into maternal circulation via exosomes.

doi: 10.1095/biolreprod.108.075481

Figure Lengend Snippet: FIG. 8. Extracellular release of miRNAs from trophoblast cells via exosomes. RNAs in exosomes enriched from the conditioned medium of EGFP-CD63-expressing BeWo cells were immunoprecipitated using sepharose 4B beads conjugated with isotype-matched nonimmune IgG (cont) or anti-CD63 (aCD63) antibody. The immunoprecipitates were then subjected to real-time RT-PCR analysis for extracellularly released MIR517A (A) and MIR21 (B). Plus sign (þ) indicates that MIR517A was exogenously introduced in EGFP-CD63-expressing BeWo cells. Saccha- romyces cerevisiae tRNA (sc-tRNA) spiked in each RNA sample was used as a control. A normalized miRNA level (miRNA/sc-tRNA) in the immunoprecipitates with anti-CD63 antibody from EGFP-CD63-express- ing BeWo cells without exogenous MIR517A is assigned a value of 1. Values are the mean 6 SD from three measurements. Significantly high levels of both endogenous and exogenous miRNAs were detected in the exosome-enriched fraction compared with those in the negative controls (*P , 0.001).

Article Snippet: Some hybridized sections were subsequently immunostained with mouse anti-CD63 monoclonal antibodies (catalog number sc-5275, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA; and catalog number H5C6, 1:400; Developmental Studies Hybridoma Bank, Iowa City, IA).

Techniques: Expressing, Immunoprecipitation, Quantitative RT-PCR, Control