The EV pellets obtained from ultracentrifugation and cell pellets were lysed with 30 µL of boiling buffer with protease inhibitor. The protein concentrations were measured using a BCA protein assay kit. A total of 100 µg of protein lysates were mixed with 6× sample buffer and boiled at 95 °C for 10 min and then loaded into a gel (Novex WedgeWell 8 to 16%, Tris-Glycine, 1.0 mm, Mini Protein Gel, 12-well, Thermo Fisher Scientific, Waltham, MA, USA). The protein was transferred into a Pierce PVDF Transfer Membrane, 0.45 µm (Thermo Fisher Scientific, Waltham, MA, USA), blocked with 5% milk (Blotting-Grade Blocker, Bio-Rad, Berkeley, CA, USA) in TBST, and then incubated in the following primary antibodies overnight at 4 °C: anti-flotillin, anti-TSG101, anti-CD9, and anti-GAPDH (Cell Signaling Technology, Danvers, MA, USA). After being washed with TBST for 10 min three times, the membranes were incubated in the AP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) for 3 h at 4 °C, washed three times, and exposed to Cytiva Amersham™ ECF™ Substrate (Thermo Fisher Scientific, Waltham, MA, USA) before visualizing the membranes using a Typhoon FLA 7000 biomolecular imager (GE Healthcare, Chicago, IL, USA).
Anti cd9
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Anti-CD9 is a laboratory reagent used for the detection and analysis of the CD9 protein. CD9 is a member of the tetraspanin family of proteins, which play important roles in cell signaling, adhesion, and migration. Anti-CD9 can be used in various analytical techniques, such as flow cytometry, immunoprecipitation, and Western blotting, to study the expression and function of CD9 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Cell Signaling Technology (5 mentions)
Anti hsp70, by Cell Signaling Technology (5 mentions)
Anti alix, by Cell Signaling Technology (5 mentions)
Anti tsg101, by Abcam (4 mentions)
Anti cd81, by Abcam (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Anti cd63, by Abcam (3 mentions)
Anti vegf, by Abcam (3 mentions)
Anti β actin, by Proteintech (3 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Anti tsg101, by Cell Signaling Technology (2 mentions)
Anti alix, by Santa Cruz Biotechnology (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Chemiluminescent detection reagent, by Zeta Life (2 mentions)
Sa00001 1, by Proteintech (2 mentions)
Anti cd81, by Cell Signaling Technology (2 mentions)
Bolt 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (2 mentions)
31 protocols using anti cd9
1
Extracellular Vesicle Protein Analysis
2
Quantifying Extracellular Vesicle Proteins
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The concentration of total protein in EV samples and cells lysed in RIPA buffer was determined using the NanoOrange™ protein quantitation kit (#N6666, ThermoFisher Scientific, Eugene, OR, USA) according to the manufacturer’s recommendations using a SpectraMax M5e microplate reader (Molecular Devices, LLC., San Jose, CA, USA). Immunoblotting was performed according to the previously described procedure [25 (link)] with the differences that 5 µg of total protein was applied to SDS-PAGE and proteins were visualized with SuperSignal™ West Femto Maximum Sensitivity Substrate (#34095, ThermoFisher Scientific, Rockford, IL, USA). The following primary and secondary antibodies and dilutions were used: anti-Alix (#sc-271975, 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Flotillin-2 (#3436S, 1:1000; Cell Signaling Technology, Topsfield, MA, USA), anti-CD9 (#13174, 1:2000; Cell Signaling Technology), anti-TSG-101 (ab125011, 1:5000; Abcam, Cambridge, UK), anti-PCNA (#sc-7907, 1:500; Santa Cruz Biotechnology), anti-Stomatin (#sc-134554, 1:500; Santa Cruz Biotechnology), anti-mouse goat polyclonal antibodies (#2367, 1:5000; Cell Signaling Technology); and anti-rabbit goat polyclonal antibodies (#29902, 1:80,000; Cell Signaling Technology).
3
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).
4
Exosomal Protein Analysis by Western Blot
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For the western blot analysis, exosomal proteins were isolated from 500 μl of plasma (the yield of exosomal protein was 9 mg/sample). The exosomal pellet was resuspended in RIPA buffer (Cell Signaling technology), supplemented with phosphatase and protease inhibitors (Roche), and incubated in ice for 20′. Samples were then harvested at 14,000 x g for 10′, and the supernatant collected in a new eppendorf. Protein concentration was determined by using Bradford Assay (Bio-Rad), following the manufacturer’s instructions. 80 μg of exosomal lysate were then loaded on a Criterion Tris-HCl 4–20% pre-cast gel (Bio-Rad), transferred onto a nitrocellulose membrane (Bio-Rad) and probed with anti-Alix (1:1000), anti-TSG101 (1:1000), anti-Calnexin (Sigma) (1:2000), and anti-CD9 (Cell Signaling Technology) (1:1000) primary antibodies, followed by isotype matched, horseradish-peroxidase-conjugated secondary antibodies. Finally, the proteins of interest were detected through chemi-luminescence reaction.
5
Western Blot Analysis of Protein Expression
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Equal amounts of protein (ranging from 20 to 100 μg) were resolved onto 10% SDS-PAGE gel and electroblotted onto PVDF membrane. Membranes were blocked with 5% BSA in Tris buffer saline supplemented with 0.1% Tween (TBST) for 1 h at RT and probed with different primary antibodies including anti-CD81 (sc-166029) and anti-SOX2 (sc-365823) from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), anti-CD9 (Cell Signaling Technology, Danvers, MA, USA, CST#13403) for overnight at 4 °C. N-MYC antibody was kindly provided by Dr. Min Kang, PharmD, TTUHSC. After washing, membranes were probed with horseradish peroxidase-conjugated secondary antibodies (rabbit/mouse, CST, Danvers, MA, USA) at RT for 1 h, and signals were detected using west pico-chemiluminescent kit (Thermo Fisher Scientific) under ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA). β-actin was used as a loading control for the protein.
6
Proteomic Analysis of Treated BMDCs
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The treated BMDCs were washed twice with precooled PBS and then lysed with a Radioimmunoprecipitation (RIPA) lysis solution containing phosphatase and protease inhibitors on ice for 30 min. The cell lysates were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany). After blocking nonspecific binding sites, the membranes were incubated with different primary antibodies [anti-14-3-3 zeta/delta, anti-enolase-1, anti-CD9, anti-NF-κB1, anti-NF-κB p65, and anti-GAPDH (Cell Signaling Technology, MA, USA)] and their respective horseradish peroxidase (HRP)-conjugated secondary antibodies. The membranes were visualized by an chemiluminescence (ECL) detection system (Merck Millipore). The intensities of the immunoreactive protein bands were analyzed with Image J (NIH, Bethesda, MD, USA).
7
Protein Expression Analysis of Extracellular Vesicles
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The protein extracts of cells (or EVs, see the “Production of EVs derived from H-DPSCs and P-DPSCs (H-EVs and P-EVs) ” section) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Then, the membranes were blocked in 5% nonfat milk in Tris-buffered saline Tween-20 (TBST, Heart). After incubating with primary antibodies including anti-β-actin (1:1000; Proteintech, Rosemont, USA; #60008-1-lg), anti-ALIX (1:1000; Cell Signaling Technology, Danvers, MA, USA; #2171), anti-HSP-70 (1:1000; Cell Signaling Technology; #4876), anti-CD9 (1:1000; Cell Signaling Technology; #13174), anti-CD81 (1:1000; Abcam, Cambridge, Britain; ab109201), anti-VEGF(1:1000; Abcam; ab46154), and anti-AngII (1:100; Santa Cruz, CA, USA; sc-74,403) at 4 °C overnight, the membranes were washed three times with TBST. Then, the cells were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000, Proteintech; SA00001-1 or SA00001-2) for 2 h at room temperature. Subsequently, blots were detected using chemiluminescent detection reagent (Zeta Life, CA, USA), and the protein bands were analyzed with ImageJ software. β-actin was employed as the housekeeping gene for internal normalization.
8
Profiling Extracellular Vesicle Proteins
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EV pellets were directly lysed in RIPA buffer (Merck KGaA, Darmstadt, Germany) with protease and phosphatase inhibitor cocktail (Merck KGaA, Darmstadt, Germany) and stored at −80 °C until use. The protein content of the purified EVs was determined by using the Bradford assay (Bio-Rad, Hercules, CA, USA). Then, protein lysate (40 μg) was loaded on a Bolt 4–12% Bis-Tris gel (Thermo Fisher Scientific, Waltham, MA, USA). Western blot analyses were performed using the following antibodies: anti-CD9 (Cell Signaling Technology, Danvers, MA, USA. Dilution: 1:1000), anti-CD81 (Cell Signaling Technology, Danvers, MA, USA. Dilution: 1:1000) primary antibodies and the corresponding anti-mouse and anti-rabbit peroxidase-linked secondary antibodies (Cell Signaling Technology, Danvers, MA, USA. Dilution: 1:5000). Signal detection was performed via chemiluminescence reaction (ECL, GE Healthcare, Chicago, IL, USA) using the MINI HD9 Western Blot Imaging System (Cleaver Scientific Ltd., Rugby, UK).
9
Immunofluorescence Staining of Tissue Sections
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The frozen tissue sections or cells were allowed to recover to room temperature and then fixed with 4% PFA for 15 min, followed by permeabilization with 0.3% Triton X-100 for 15 min. Afterward, nonspecific antigen was blocked with 3% BSA for 1 h. Primary antibodies were added to the tissue and incubated at 4 °C overnight. On the following day, fluorescence-labeled secondary antibodies (Alexa Fluor 488 or tetramethylrhodamine conjugated, ZSGB-BIO, Beijing, China) were applied for 1 h at 37 °C in the dark. Finally, DAPI dye was used for nuclear counterstaining. All the primary antibodies were as follows: anti-Ki67 (28074-1-AP, Proteintech, Wuhan, China), anti-CASPASE3 (19677-1-AP, Proteintech, Wuhan, China), anti-P-MAPK (#4511, Cell Signaling Technology, Boston, USA), anti-KISS-1 (sc-18134, Santa Cruz, USA), anti-TER119 (sc-19592, Santa Cruz, USA), anti-SREBP2 (ab30682, Abcam, Cambridge, MA, UK), and anti-CD9 (#98327 S, Cell Signaling Technology, Boston, USA). All images were captured on a Leica confocal microscope (Weztlar, Germany).
10
Exosome Protein Analysis by Immunoblotting
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