Proteins in exosomes or cells were lysed and extracted using a whole-protein extraction kit (KeyGEN, Nanjing, China). Then, the proteins were separated by SDS–PAGE and transferred to NC membranes, blocked in 5% skim milk at room temperature for 1.5 h, and incubated with the following primary antibodies: anti-CD9 (1:300; Abcam, Cambridge, MA, USA), anti-CD63 (1:300; Abcam, Cambridge, MA, USA), anti-TSG101 (1:300; Abcam, Cambridge, MA, USA), anti-calnexin (1:300, Abcam, Cambridge, MA, USA), anti-TNFα (1:1,000; Abcam, Cambridge, MA, USA), anti-IL-6 (1:1000; Abcam, Cambridge, MA, USA), anti-ALP (1:1,000, Boaosen, China), anti-RUNX2 (1:1,000; Abcam, Cambridge, MA, USA), anti-COL-1 (1:1,000; Abcam, Cambridge, MA, USA), and anti-GAPDH (1:5,000; Abcam, Cambridge, MA, USA). The blot was then stained with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (KGAA35; KeyGEN, Nanjing, China). Finally, the protein expression level was detected using a chemiluminescence detection system. Each experiment was repeated 3 times.
Anti cd9
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Anti-CD9 is a monoclonal antibody that recognizes the cell surface protein CD9. CD9 is a member of the tetraspanin family of proteins and is involved in various cellular processes including cell adhesion, migration, and signaling.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd63, by Abcam (54 mentions)
Anti tsg101, by Abcam (40 mentions)
Anti cd81, by Abcam (24 mentions)
Pvdf membrane, by Merck Group (19 mentions)
Anti calnexin, by Abcam (15 mentions)
Anti gapdh, by Abcam (12 mentions)
Anti alix, by Abcam (12 mentions)
Bca protein assay kit, by Beyotime (7 mentions)
Anti β actin, by Abcam (7 mentions)
Anti cd81, by Santa Cruz Biotechnology (7 mentions)
Anti gm130, by Abcam (6 mentions)
Anti akt, by Abcam (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Anti gapdh, by Proteintech (6 mentions)
Anti alix, by Cell Signaling Technology (4 mentions)
Whatman, by Cytiva (4 mentions)
Anti cd63, by Santa Cruz Biotechnology (4 mentions)
Anti β actin, by Cell Signaling Technology (4 mentions)
Anti p akt, by Cell Signaling Technology (4 mentions)
Anti gm130, by BD (4 mentions)
110 protocols using anti cd9
1
Exosomal Protein Profile Analysis
2
Protein Extraction and Western Blot Analysis
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Total proteins were extracted using cell lysis buffer (BioFeng, Changsha, Hunan, China). After the protein concentrations were determined by BCA kits, the protein samples were extracted and separated by 10% SDS-PAGE gel and then transferred to a PVDF membrane (Millipore, USA). The membrane was then blocked with 5% skimmed milk and incubated overnight, using the following main detection antibodies at 4°C: anti-GAPDH (1 : 1,000; Abcam), anti-Histon-H3 (1 : 1,000; Abcam), anti-Hsp60 (1 : 1,000; Abcam), anti-cGAS (1 : 1,000; Abcam), anti-STING (1 : 1,000; Abcam), anti-pSTING (1 : 1,000; Abcam), anti-TBK1 (1 : 1,000; Abcam), anti-pTBK1 (1 : 1,000; Abcam), anti-IRF3 (1 : 1,000; Abcam), anti-pIRF3 (1 : 1,000; Abcam), anti-PD-L1 (1 : 1,000; Abcam), anti-CD9 (1 : 1,000; Abcam), anti-TSG101 (1 : 1,000; Abcam), anti-Alix (1 : 1,000; Abcam), anti-Hsp90 (1 : 1,000; Abcam), or anti-β-actin (1 : 5,000; Proteintech). We washed 3 times with TBS-T, and the membranes were cultured with the secondary antibody at 24°C for 1 hr. The western blots were pictured using an ECL Reagent (Pierce, USA), and the density was verified using ImageJ software (NIH, USA).
3
Extracellular Vesicle Protein Characterization
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After total protein concentration was determined, AT-EVs—amounting to 30 μg protein—were loaded onto a 10%–15% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and electrophoresed. AT-EV proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes, incubated with primary anti-CD9 (1:1,000, #ab236630), anti-CD63 (1:1,000, #ab134045), anti-CD81 (1:1,000, #ab109201), anti-TSG101 (1:1,000, #ab125011), and anti-GM130 (1:1,000, #ab52649) antibodies (all obtained from Abcam, Cambridge, UK), followed by incubation with secondary antibodies (1:5,000, #111-035-045, Jackson ImmunoResearch, West Grove, PA, United States). Protein expression was evaluated using a BeyoECL Plus kit (#MA0186, Meilunbio, Dalian, Liaoning, China).
4
EV Protein Marker Analysis via Immunoblotting
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EVs or tissue samples were analyzed for the content of EV protein markers using immunoblotting. The protein concentration was measured using a BCA Protein Assay Kit (Servicebio, Wuhan, China) according to the manufacturer’s instructions. Antibodies used for immunostaining were anti-CD9 (Abcam, Cambridge, UK), anti-ALIX (Cell Signaling Technology, Danvers, MA, USA), and anti-TSG101 (Abclonal, Woburn, MA, USA).
5
Immunofluorescence analysis of Cdc42 in cells
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Anti-CD9, CD63, Alix, calnexin, CD31, CD34, CD45, CD73, CD90 and CD105 antibodies were purchased from Abcam (Cambridge, UK). Anti-Ki-67, RhoA, Rac1, Cdc42 and β-actin antibodies were purchased from Cell Signaling Technology (Danvers, USA). Alexa Fluor 488 and Alexa Fluor 568 secondary antibodies were purchased from Proteintech (Rosemont, IL, USA). Goat anti-rabbit/anti-mouse IgG IRDyel 800cw secondary antibodies were purchased from Abbkine (Redlands, CA, USA). PKH-26 and PKH-67 kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine TM RNAiMAX, FM TM 4-64FX and ActinGreen TM 488 were purchased from Thermo Fisher (Eugene, Dregon, USA). SiCdc42, Cdc42-EGFP and Cdc42-mCherry fusion protein expression plasmids were purchased from GenePharma (Suzhou, China). Cdc42 inhibitor ML141 was purchased from MedChemExpress (Monmouth Junction, USA).
6
Extracellular Vesicle Protein Profiling
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Total proteins were extracted from HUVEC employing radio immunoprecipitation assay lysis buffer (cat. no. AS1004) obtained from Aspen Pharmacare Holdings Ltd. Cell lysates (1 × 104) were treated with 10% SDS‐PAGE and the bicinchoninic acid method was employed to estimate protein concentration. Proteins (50 µg) were then placed onto a 10% SDS‐PVDF membrane. Bovine serum albumin (5%, Abcam) was used to block the PVDF membrane at room temperature for 2 hours. Proteins were then visualized using a chemiluminescent detection system (Canon, Inc; cat. no. LiDE110). Following antibodies were used in the Western blotting analysis: anti‐TSG 101 (1:1000; cat. no. Ab125011; Abcam), anti‐CD9 (1:1000; cat. no. Ab92726; Abcam), anti‐PIK3R3 (1:500; cat. no. Ab238509; Abcam), anti‐cyclin D1 (1:1000; cat. no. Ab40754; Abcam), anti‐cyclin D3 (1:1000; cat. no. Ab112034; Abcam) and anti‐GAPDH (1:10 000; cat no. ab37168; Abcam). Measurements were made in triplicate for all experiments.
7
Isolation and Characterization of Extracellular Vesicles
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ExoQuick-TC (System Biosciences, EXOTC50A-1, California, USA), BCA protein assay kit (New Cell & Molecular Biotech Co., Ltd, WB6501, Suzhou, China), PLGA (Jinan Daigang Biomaterial Co., Ltd., Jinan, China), 5×SDS protein loading buffer (Beyotime, P0015L, Shanghai, China), PVDF membrane (Millipore, ISEQ00010/IPVH00010, USA), DII (Beyotime, C1036, Shanghai, China), DIO (Beyotime, C1038, Shanghai, China), DAPI (Biosharp, 28718-90-3, Anhui, China), Cell Counting Kit-8(MedChemExpress, HY-K0301, USA), Calcein-AM/PI Double Stain Kit (Yeasen, 40747ES76, Shanghai, China), BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, C3206, Shanghai,China), RANKL (R&D, 462-TEC-010/CF, Minnesota, USA), DIR dye-labeled (Yeasen, 40726ES10, Shanghai, China), anti-CD9 (Abcam, ab236630, UK), anti-CD63(Abcam, ab134045, UK), anti-TSG101 (Abcam, ab125011, UK), anti-Calnexin (Abcam, ab22595, UK), anti-OCN(Abcam, ab93876, UK), anti-IL-6 (Abcam, ab290735, UK), and anti-TNF-α (Abcam, ab1793, UK).
8
Comprehensive Western Blot Analysis of Key Proteins
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The specific primary antibodies involved in western blot(WB) analysis were as follows: anti- VE-cadherin (1:1000), anti-ZO-1 (1:1000), anti-TSG101(1:1000, Abcam, UK), anti-VEGFR-2 (1:1000, Abcam, UK), anti-Fas (1:1000, Abcam, UK), anti-p53 (1:1000), anti-HIPK3 (1:1000), anti-Caspase-3(1:1000, Abcam, UK), anti-HNRNPA1(1:1000), anti-HIF-1α(1:1000), anti-DDDDK tag(1:1000), anti-CD9 (1:1000, Abcam, UK), anti-CD63(1:1000, Abcam, UK), anti-ALIX(1:1000, Abcam, UK), anti-β-actin(1:1000, Proteintech, Wuhan, China), anti-SFRS2(1:1000, Abcam, UK), anti-ACO-1(1:1000, Abcam, UK). The secondary antibody was HRP-IgG (1:10000, Proteintech, Wuhan, China) and β-actin was used as internal reference.
9
Purification and Characterization of TIMP2-Enriched Exosomes
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Exosomes were purified from TIMP2-transduced hucMSC- (huc-exoTIMP2-) conditioned media by using ultracentrifugation (Beckman Coulter, Brea, CA, US). Briefly, the hucMSCsTIMP2 were cultured till 80% confluency, washed with PBS, and reseeded in DMEM/F12 (HyClone, GE Healthcare, Waukesha, WI, US) including 10% exosome-free FBS (centrifuged at 100,000g for 8 hrs to eliminate preexisting bovine-derived exosomes) and 1% PS. After 24 hrs, the conditioned medium was centrifuged for 10 min at 2,000g to remove cell debris. The huc-exoTIMP2 were purified from the supernatant by ultracentrifugation at 100,000 g for 70 min. All procedures were carried out at 4°C. Exosomes were finally filtered through 0.22 μm Whatman polycarbonate filters (Whatman, Maidstone, UK) and stored at -80°C. hucMSCs with lentivirus-NC were subjected to the same protocol to obtain the huc-exoNC.
The Hitachi H-7650 transmission electron microscope (Hitachi, Tokyo, Japan) was used to observe the exosomal morphology of both huc-exoNC and huc-exoTIMP2 directly. The BCA protein assay kit (Beyotime, Shanghai, China) was used to measure the protein concentration of exosomes and characterized by Western blot with anti-CD9 and anti-CD63 antibodies (all from Abcam, Cambridge, MA, US).
The Hitachi H-7650 transmission electron microscope (Hitachi, Tokyo, Japan) was used to observe the exosomal morphology of both huc-exoNC and huc-exoTIMP2 directly. The BCA protein assay kit (Beyotime, Shanghai, China) was used to measure the protein concentration of exosomes and characterized by Western blot with anti-CD9 and anti-CD63 antibodies (all from Abcam, Cambridge, MA, US).
10
Western Blot Analysis of Intestinal Proteins
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Intestinal frozen tissues were thawed at 4°C, and protein was extracted with RIPA buffer (Biyuntian, China) according to the manufacturer's instructions. Nuclear protein from NCM460 cells was obtained by sonication of nuclear pellets followed by centrifugation. Equal amounts of protein were loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. After electrophoresis, polyvinylidene uoride (PVDF) membranes were blocked with 5% nonfat dry milk in TBS-T (20 mM Tris/HCl pH 7.2, 150 mM NaCl, and 0.1% Tween-20) and incubated overnight with different primary antibodies. The dilution concentration was as follows: anti-CD9 (1:500, Abcam, USA), anti-Occludin (1:500, Thermo Scienti c, USA), anti-ZO-1 (1:125, Thermo Scienti c, USA), anti-E-cadherin (1:1000, Cell Signaling Technology, USA), and anti-TMIGD1 (1:200, Proteintech, China). Next, the membranes were incubated with peroxidase-conjugated anti-mouse IgG (1:2,500; Thermo Scienti c, USA) or anti-rabbit IgG (1:5,000; Thermo Scienti c, USA) followed by treatment with Super Signal West Pico chemiluminescent substrate (Thermo Scienti c, USA). Protein bands were detected by a LAS-3000 image analyzer (Fuji lm, Barcelona, Spain), and the protein levels were quanti ed by densitometry using Image Gauge Version 4.0 software (Fuji lm). Data were normalized to β-actin.
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