eEV and eEV-IL-3 were lysed in lysis buffer (RIPA buffer with proteinase inhibitors). Starting from the same EV particle number, protein samples were quantified by the Bradford method before performing Western blot (50 µg proteins/each sample were loaded). Anti-CD63, anti-CD81, antiHSP90, anti-GM130, anti-Bcl-2 (Abcam, Milan, Italy), anti-MEK1/2, (Cell Signaling, Danvers, MA, USA), anti-CD29, anti-caveolin-1, anti-p-eNOS-ser1177 (Invitrogen, Carlsbad, CA, USA), and anti-vinculin (Millipore, Milan, Italy) antibodies were used as primary antibodies. Appropriate HRP-conjugated secondary antibodies (BioRad, Milan, Italy) were used, and proteins were detected with Clarity Western ECL substrate (BioRad, Milan, Italy). Image Lab Software (BioRad Milan, Italy) instrument was used for densitometric analysis. Data are expressed as arbitrary unit. Ponceaus-staining has been used as input (EV protein content normalization).
Anti cd63
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Anti-CD63 is a primary antibody used to detect the CD63 antigen, a glycoprotein that is a member of the tetraspanin family. CD63 is expressed on the surface of various cell types, including platelets, monocytes, and activated T cells. The Anti-CD63 antibody can be used in techniques such as flow cytometry, immunohistochemistry, and western blotting to study the expression and localization of the CD63 antigen.
Automatically generated - may contain errors
Lab products found in correlation
Anti tsg101, by Abcam (62 mentions)
Anti cd9, by Abcam (54 mentions)
Anti cd81, by Abcam (35 mentions)
Pvdf membrane, by Merck Group (23 mentions)
Anti alix, by Abcam (20 mentions)
Anti calnexin, by Abcam (20 mentions)
Anti gapdh, by Abcam (15 mentions)
Anti gm130, by Abcam (13 mentions)
Anti β actin, by Abcam (12 mentions)
Anti tsg101, by Santa Cruz Biotechnology (11 mentions)
Anti akt, by Abcam (10 mentions)
Anti hsp70, by Abcam (9 mentions)
Bca protein assay kit, by Beyotime (8 mentions)
Anti β actin, by Cell Signaling Technology (7 mentions)
Ripa lysis buffer, by Beyotime (7 mentions)
Anti α sma, by Abcam (7 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Anti vimentin, by Abcam (6 mentions)
Anti gapdh, by Santa Cruz Biotechnology (6 mentions)
Anti gapdh, by Proteintech (5 mentions)
186 protocols using anti cd63
1
Extracellular Vesicle Protein Profiling
2
Exosome Characterization by TEM and DLS
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Exosome morphology was observed using TEM. The exosome suspension was mixed with an equal volume of 4% paraformaldehyde, and 10 μL of the mixture was placed on a clean copper grid (RT) at room temperature. Uranyl acetate staining was negative. The images were acquired by observation with a JEOL 1200EX TEMSCAN microscope. The exosomal suspensions were analyzed for particle size using dynamic light scattering (DLS) (Nanosizer ™ instrument, Malvern Instruments, Malvern, UK).
The extracted exosomes were resuspended in cell lysate (Beyotime, Nantong, China) supplemented with 1% PMSF. The protein concentration of exosomes was determined using a Pierce BCA protein detection kit (Thermo Fisher Scientific, Rockford, IL, USA). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12%) were prepared with 20 μg of protein on each sample. Anti-CD63, anti-TSG101, anti-β-actin, and anti-GAPDH were purchased from Abcam (Cambridge, UK). All antibodies used in western blot are diluted 1:1000.
The extracted exosomes were resuspended in cell lysate (Beyotime, Nantong, China) supplemented with 1% PMSF. The protein concentration of exosomes was determined using a Pierce BCA protein detection kit (Thermo Fisher Scientific, Rockford, IL, USA). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12%) were prepared with 20 μg of protein on each sample. Anti-CD63, anti-TSG101, anti-β-actin, and anti-GAPDH were purchased from Abcam (Cambridge, UK). All antibodies used in western blot are diluted 1:1000.
3
Exosome Characterization and Nrf-1 Immunoblotting
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The cell pellet and exosomes were homogenized in RIPA buffer. The homogenates were centrifuged, the supernatant was isolated, and immunoblotting was performed using exosomal positive and negative markers, including anti-CD63 (Abcam), anti-TSG101 (Abcam) and anti-β-actin (Cell Signaling Technology) to confirm the success of exosome extraction. The conditioned medium recovered after the exosome isolation procedure was used as a negative control. Immunoblot analysis was performed for Nrf-1 (Abcam) and band densities were normalized to GAPDH (Millipore Sigma).
4
Exosome Regulation by miR-92a-3p
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LPS, GW4869 and PKH-67 were purchased from Sigma-Aldrich. The following antibodies were used: anti-CD68, anti-CD9, anti-CD63, anti-PTEN, anti-Alix, anti-Akt, anti-p-Akt, goat Anti-Rabbit IgG H&L (Alexa Fluor® 555) (Abcam), anti-p65, and anti-p-p65(Beyotime); miR-92a-3p mimic, mir-92a-3p inhibitor, control RNAs and the primers for miRNAs were all purchased from Guangzhou RiboBio.
5
Comprehensive Protein Expression Profiling of Extracellular Vesicles
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Protein extracts were boiled in RIPA buffer (Beyotime) and separated by sodium dodecyl sulfate–polyacrylamide electrophoresis gel electrophoresis (SDS‐PAGE). The proteins were then transferred to polyvinylidene fluoride membranes (Millipore) and probed with anti‐CD9, anti‐CD63, anti‐ALIX, anti‐TSG101, anti‐EGFR, anti‐FXR1, anti‐TLR‐4, anti‐MMP2, anti‐TGF‐β1, anti‐phospho‐STAT5, anti‐STAT5, anti‐phospho‐ERK1/2, anti‐ERK1/2, anti‐phospho‐AKT, anti‐AKT, anti‐Ki‐67, anti‐PTEN (Abcam), anti‐Bcl‐2, anti‐Bax, anti‐E‐Cadherin, anti‐Vimentin, and anti‐GAPDH antibodies (Cell Signaling Technology). Electrochemiluminescence (Millipore) was applied to determine protein expression levels.
6
Inhibition of Exosomal Secretion in Cancer
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The exosomal secretion inhibitor GW4869 was obtained from Sigma-Aldrich (St. Louis, MO, USA). C-A1 was bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and fedratinib was obtained from MedChemExpress (Monmouth Junction, NJ, USA). The primary antibodies used for western blot and immunofluorescence assay in our study included anti-LCP1 (Cell Signaling Technology [CST], USA; Proteintech, China), anti-Nrdp1 (Santa Cruz Biotechnology), anti-hemagglutinin (HA; CST), anti-Ki-67 (CST), anti-c-Myc (CST), anti-CDK4 (CST), anti-cyclin D1 (CST), anti-N-cadherin (CST), anti-E-cadherin (CST), anti-vimentin (CST), anti-MMP-2 (CST), anti-JAK2 (CST), anti-phospho-JAK2 (CST), anti-STAT3 (CST), anti-phospho-STAT3 (CST), anti-CD63 (Abcam, UK), anti-CD9 (CST), anti-calnexin (CST), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; CST), and anti-Lamin B1 (CST). The horseradish peroxidase (HRP)- and fluor-conjugated secondary antibodies (Jackson ImmunoResearch, USA) were utilized for western blot and immunofluorescence assay, respectively.
7
Western Blot Analysis of Exosomal Proteins
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Cell or exosome lysis was performed in RIPA lysis buffer with complete proteolytic and phosphatase inhibitors (Sigma). A total of 40 μg of protein per well was loaded onto SDS‐polyacrylamide gels and next transferred onto polyvinylidene difluoride membranes (PVDF; Millipore). Thereafter, membranes were blocked with 5% skimmed dry milk and then incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase‐conjugated secondary antibody for 1 h at room temperature. Finally, the membranes were analyzed using an automatic chemiluminescence imaging analysis system (Tanon). The primary antibodies anti‐CD9 (CST), anti‐CD63 (Abcam), anti‐SOCS3 (R&D systems), anti‐HIF1α (CST), anti‐p‐AKT (CST), anti‐AKT (CST), anti‐p‐STAT3 (CST), anti‐STAT3 (CST), and anti‐GAPDH (Proteintech) were used in the experiments.
8
Identification of iMSCs-Exo Markers
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Western blotting was used to identify iMSCs-Exo markers CD63, CD81, and CD9 [33 (link)]. Briefly, 5 × protein-loading buffer was added directly to the iMSCs-Exo sample and heated at 95°C for 5 minutes. Next, iMSCs-Exo protein was loaded and resolved in 12% SDS-PAGE polyacrylamide gels. The protein sample was run at 120 V for 45 minutes and transferred onto nitrocellulose membranes (Whatman, Maidstone, Kent, UK) for 1.5 hours at 100 mA. The presence of CD63, CD81, and CD9 was assessed by exposing the membranes to primary rabbit polyclonal anti-CD63 (1:1,000), anti-CD81 (1:1,000), and anti-CD9 (1:1,000) (Abcam, Cambridge, UK). The membranes were washed three times in 1 × Tris-buffered saline with tween (TBST) for 5 minutes and incubated for 1 hour in TBST containing horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Abcam). Proteins were detected by using enhanced chemiluminescence (Thermo Fisher) and imaged by using an Image Quant LAS 4000 mini bio-molecular imager (GE Healthcare, Uppsala, Sweden).
9
Exosomal Protein Verification by Western Blot
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To verify the isolation of exosomes from the serum, western blot analysis for CD9 and CD63, which are enriched in exosomes, was performed. Total proteins were extracted using lysis buffer (Pro-Prep, iNtRON Biotechnology, South Korea) and 20 μg of protein was separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (GE Health care, Piscataway, NJ). After blocking with 5% skimmed milk for 1 hour at room temperature, membranes were incubated overnight at 4°C with primary antibody (anti-β-actin 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CD9 1:1000 (Cell Signaling, Danvers, MA, USA) or anti-CD63 1:1000 (Abcam, Cambridge, UK)) followed by horseradish peroxidase-conjugated anti-mouse 1:1000 or anti-rabbit secondary antibody 1:1000 (Novus Biologicals, Littleton, CO, USA), and incubated for 1 hour at room temperature. After incubation, membranes were washed and proteins revealed by Western Blotting Luminol Reagent (Bio-Rad, Hercules, CA, USA).
10
Proteomics Analysis of EV Proteins
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Proteins were extracted from platelets or endothelial-derived EVs, separated on 10–12% sodium dodecyl sulfate–polyacrylamide gels, and transferred onto PVDF membranes, which were soaked in Tris-buffered saline–Tween (TBST) solution containing 5% bovine serum albumin (room temperature) to block non-specific binding. Subsequently, the membranes were exposed to the primary antibodies: anti-PDI (Abcam), anti-ERP57 (Abcam), anti-ERP46 (Immunoway, Plano, TX, USA), anti-GRP94 (Abcam), anti-CD63 (Abcam), anti-Calnexin (Abcam), and anti-TSG101 (Abcam). After staining overnight at 4 °C, the membranes were washed three times with TBST and incubated with horseradish peroxidase (HRP)-labeled anti-rabbit (ZSGB-BIO, Beijing, China) or anti-mouse (ZSGB-BIO) secondary antibodies at room temperature for 90 min. After washing thrice more with TBST, an enhanced chemiluminescence (ECL) reagent was added, and images were acquired and analyzed quantitatively using Image J.
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