The sEVs were lysed in a reagent containing a cocktail of protease inhibitors to extract proteins. These proteins were subjected to Western blotting assay using standard procedures. Briefly, after resolution by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), proteins were electrotransferred to a cellulose nitrate membrane (Millipore), which was subsequently incubated at 4°C overnight with either mouse anti-CD63 (1:2000, Abcam), mouse anti-CD9, mouse anti-Tsg101, mouse anti-Alix (all diluted to 1:2,000, Abcam), or mouse anti-Calnexin (1:3,000, Abcam). The complex was then incubated with IRDye 680 secondary antibody, anti-rabbit, or anti-mouse for 1 h at normal laboratory temperature and then characterized by Odyssey infrared imaging system (LI-COR Biosciences).
Mouse anti cd63
Manufactured by Abcam
Sourced in United States, United Kingdom
Mouse anti-CD63 is a primary antibody that binds to the CD63 antigen, which is a membrane glycoprotein expressed on the surface of various cell types, including endosomes, lysosomes, and exosomes. This antibody can be used for the detection and analysis of CD63 expression in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti tsg101, by Abcam (5 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Mouse anti cd9, by Abcam (3 mentions)
Mouse anti β actin, by Merck Group (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (3 mentions)
Rabbit anti cd9, by Abcam (3 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
Amersham imager, by Cytiva (2 mentions)
Mouse anti β actin, by Abcam (2 mentions)
Mouse anti eea1, by BD (2 mentions)
Mouse anti α tubulin, by Santa Cruz Biotechnology (2 mentions)
Goat anti vegfr2, by R&D Systems (2 mentions)
Rabbit anti alix, by Abcam (2 mentions)
Rabbit anti alpha tubulin, by Abcam (2 mentions)
Rabbit anti hsp70, by Abcam (2 mentions)
Cellulose nitrate membrane, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Mouse anti alix, by Abcam (1 mentions)
Mouse anti calnexin, by Abcam (1 mentions)
26 protocols using mouse anti cd63
1
Extracellular Vesicle Protein Profiling
2
Comprehensive Proteomic Analysis of Extracellular Vesicles
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Cells and EV proteins were separated on 10%–15% SuperSep™ Ace gels (Fujifilm) and transferred onto polyvinylidene difluoride membranes using a Trans‐Blot® Turbo™ Transfer System (Bio‐Rad). Western blotting was performed on iBind™ Western Systems (Thermo Fisher Scientific) according to the manufacture's instructions14 using the following primary antibodies: mouse anti‐αSMA (Sigma, A5228), rabbit anti‐FAPα (Abcam, ab137549), rabbit anti‐E‐cad (Cell Signaling Technologies, 24E10), mouse anti‐CD9 (Santa Cruz, sc‐59,140), mouse anti‐CD63 (Abcam, ab59479), and mouse anti‐CD81 (Santa Cruz, sc‐166,029). Protein bands were visualized using HRP‐conjugated secondary antibodies and Chemi‐Lumi One (Nacalai Tesque) on an Amersham Imager 600 (GE Healthcare).
3
Characterization of Extracellular Vesicles by WB
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The following anti-human antibodies and their dilution rates were used for western blot (WB) analyses: mouse anti-CD63 (Abcam, Cambridge, MA, USA; 1:1,000), mouse anti-CD133 (Abnova Corp., Taipei city, Taiwan; 1:1,000), rabbit anti-aquaporin 5 (AQP5) (Abcam; 1:500), mouse anti-β-actin (Sigma–Aldrich, St. Louis, MO, USA; 1:1,000), rabbit anti-CD26 (Abcam; 1:1,000), mouse anti-CD81 (EXBIO, Plaha, Czech; 1:1,000), mouse anti-EpCAM/TROP-1 (R&D Systems, Minneapolis, MN, USA; 1:1,000), rabbit anti-HSP70 (System Biosciences, Mountain View, CA, USA; 1:1,000), mouse anti-TSG101 (BD Transduction Laboratories, San Jose, CA, USA; 1:500), rabbit anti-CD44 (Sigma–Aldrich; 1:1,000), rabbit anti-CD24 (Santa Cruz Biotechnology, Dallas, TX, USA; 1:100) and mouse anti-CD9 (Abcam; 1:500). Secondary antibodies coupled to horseradish peroxidase were as follows: goat anti-rabbit IgG (H+L)-HRP conjugate (Bio-Rad, Hercules, CA, USA; 1:2,000), goat anti-mouse IgG [(H+L)-HRP conjugate (Bio-Rad; 1:2,000)] and rabbit anti-goat IgG (H+L)-HRP conjugate (Bio-Rad; 1:2,000). Iohexol (Nycodenz) and iodixanol (OptiPrep) purchased from Axis-Shield PoC (Oslo, Norway) were used as density gradient media. Each gradient medium was prepared in 0.02 M HEPES [4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid]/NaOH, pH 7.2 buffer.
4
Exosome Characterization by SEM
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Scanning electron microscopy (SEM) was applied to examine the isolated exosomes, which wereloaded onto a carbon-coated electron microscopy grid, as described previously.15 After fixing with 2% glutaraldehyde and 2% paraformaldehyde in 0.1 mol/L sodium cacodylate buffer at pH 7.3 for 3 hrs at room temperature, the samples were critical point dried, mounted on specimen slides, sputter-coated, and visualized using a Hitachi S3400 scanning electron microscope. Exosome proteins were subjected to electrophoresis and transferred to PVDF membranes. The membranes were probed with mouse anti-CD63 (1:200, Abcam, Cambridge, UK). The details of this experimental process and its effectiveness are shown in our previous studies.12 (link)
5
Extracellular Vesicle Characterization by Flow Cytometry
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AB, SMV, or EXO samples were adsorbed onto 4-mm aldehyde/sulphate latex beads (Invitrogen, Paisley, UK) overnight at 4°C. The reaction was stopped by adding glycine 100 mM. Membrane-bound beads were washed in PBS/1% BSA, incubated with mouse anti-CD63 (Abcam, Cambridge, UK) or appropriate isotype control for 30 min at 4°C, stained with FITC-conjugated secondary antibody (R&D Systems) for 30 min at 4°C, and resuspended in 0.5 ml of PBS. Samples were analyzed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA).
6
Exosomal Protein Characterization by Western Blot
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Exosomal protein content was measured using a BCA assay. Total protein (40 μg) was electrophoresed using 10% SDS–PAGE, and transferred to PVDF membranes (Millipore, USA). Membranes were blocked for 1 h at RT with 5% non-fat milk. Then membranes were incubated overnight at 4 °C with the primary antibody (rabbit anti-CD29, 1:1000, Proteintech or mouse anti-CD63, 1:1000, Abcam) diluted in TBST with 5% BSA. Membranes were washed with TBST, followed by incubation for 1 h at room temperature with either horseradish-peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies or HRP-conjugated goat anti-mouse (1:1000, Beyotime). The bands were visualized using ECL detection.
7
Western Blot Analysis of Cellular Markers
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Western blotting was performed using standard procedures. Briefly, protein samples (25mg) were separated on polyacrylamide-SDS gels and electrophoretically transferred to polyvinylidene difluoride membranes (Millipore). Subsequently, membranes were blocked and incubated with mouse anti-CD81 (1:1000, Abcam), mouse anti-CD63 (1:1000, Abcam), mouse anti-HIF-1α (1:1500, ProteinTech Group, Inc.), mouse HIF-2α (1:1500, ProteinTech Group, Inc.), rabbit anti-PTEN (1500, Abcam), rabbit anti-E-cadherin (1:1000, Abcam), mouse anti-vimentin (1:1000, Abcam), rabbit anti-mTOR (1:1000, Abcam), rabbit anti-AKT (1:1000, Abcam), rabbit anti-pAKT (1:1000, Abcam), and rabbit anti-tubulin (1:1000 Abcam) antibodies. Anti-rabbit or anti-mouse IgG was used as the secondary antibody (1:8000). The signal intensity was evaluated using Quantity One 4.4.0 software.
8
Antibodies for HSV-1 Protein Detection
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The following primary antibodies were used for Western blotting and immunofluorescence and were kindly provided: mouse anti-gD (LP14) and mouse anti-gB (R69), from Tony Minson (University of Cambridge); mouse anti-VP16 (LP1), from Colin Crump (University of Cambridge); mouse anti-gE, from David Johnson (Oregon Health and Science University); and mouse anti-nectin1 (CK6), from Claude Krummenacher (Rowan University, NJ). Our rabbit VP22-specific antibody (AGV031) was described previously (68 (link)). Commercially available antibodies used in this study included mouse anti-V5, mouse anti-gC, mouse anti-β-actin, mouse anti-CD63, rabbit anti-giantin, mouse anti-CHMP4A, and rabbit anti-CHMP4B (all from Abcam); mouse anti-CD71 (Santa Cruz); and mouse anti-γ-tubulin and mouse anti-α-tubulin (Sigma). Goat anti-mouse IRDye 680RD and goat anti-rabbit IRDye 800CW (Li-Cor Biosciences) secondary antibodies were used as appropriate for Western blotting, while Alexa Fluor-conjugated secondary antibodies (Invitrogen) were used for immunofluorescence. Endocytic structures were labeled by incubating cells with Texas Red-conjugated transferrin (Invitrogen) or Alexa Fluor 488-conjugated transferrin (Molecular Probes) at a concentration of 1 μg/ml for 30 min. Brefeldin A (Sigma) was used at a concentration of 1 μg/ml.
9
HUVEC Isolation and Characterization
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Human umbilical vein endothelial cells (HUVECs) were isolated and cultured as previously described 30 , 31 . The following reagents were purchased: goat anti‐VEGFR2 (R&D Systems), rabbit anti‐phospho‐VEGFR2 (Y1175), rabbit antibodies to native and phosphorylated c‐Akt (S473), PLCγ1 (Y783) and p38 (T180/Y182), rabbit anti‐ERK1/2, mouse anti‐phospho‐ERK1/2 (T202, Y204), rabbit anti‐USP8 (Cell Signaling Technologies), rabbit anti‐TGN46, mouse anti‐EEA1 (BD Biosciences), mouse anti‐CD63 (AbCam), mouse anti‐α‐tubulin, mouse anti‐PECAM1, mouse anti‐transferrin receptor (TfR) (Santa Cruz Biotechnology), mouse FK2 anti‐ubiquitin (Affiniti Research Products), rabbit Apu2 anti‐K48‐linked ubiquitin (Millipore), rabbit Apu3 anti‐K63‐linked ubiquitin (Millipore), HRP‐conjugated secondary antibodies (PerBio Sciences), AlexaFluor‐conjugated secondary antibodies (Invitrogen), endothelial cell growth medium (ECGM) (PromoCell), non‐targeting and USP8 siGENOME SMARTpool siRNA duplexes (Dharmacon, GE Healthcare) and recombinant human VEGF‐A165a (Genetech Inc.).
10
Characterizing VEGFR2 Signaling in Endothelial Cells
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Primary HUVECs were cultured as previously described (Fearnley et al., 2014 (link); Howell et al., 2004 (link)), HDMECs and appropriate growth media were from PromoCell (Heidelberg, Germany). Purified primary and secondary antibodies were typically used at 1 µg/ml for microscopy and at 0.1 µg/ml for immunoblotting. These antibodies were goat anti-VEGFR2 (R&D Systems, Minneapolis, USA), rabbit anti-phospho-VEGFR2 (Y1175), rabbit anti-UBA1 (Cell Signaling Technologies, Danvers, USA), rabbit antibodies to native and phosphorylated PLCγ1 (Y783), rabbit anti-ERK1/2, mouse anti-phospho-ERK1/2 (T202, Y204), mouse anti-α-tubulin (Santa Cruz Biotechnology, USA), mouse anti-transferrin receptor (TfR), mouse FK2 anti-ubiquitin (Affiniti Research Products, Exeter, UK), mouse anti-EEA1 (BD Biosciences, California, USA), mouse anti-CD63 (Abcam, Cambridge, UK), mouse anti-LAMP2 (Santa Cruz, USA), HRP-conjugated secondary antibodies (Thermo Fisher, Loughborough, UK) and Alexa Fluor-conjugated secondary antibodies (ThermoFisher). Endothelial cell growth medium (PromoCell), non-targeting and UBA1 siRNA duplexes (GE Dharmacon, UK) and recombinant human VEGF-A165 (Genentech Inc., San Francisco, USA) were obtained as stated. Chemicals were obtained from Sigma-Aldrich (Poole, UK) or Thermo Fisher (Loughborough, UK).
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