The primary antibodies used were anti-collagen I, anti-VEGF and anti-PDGFBB (Abcam, Cambridge, UK); anti-TSG101, anti-CD9, anti-CD63, and anti-CD81 (System Biosciences, Mountain View, CA, USA). The primary antibodies anti-CD41, anti-TGF-β, anti-bFGF, anti-cleaved-caspase-3, anti-Runx2, anti-β-catenin, anti-β-tubulin, anti-calnexin, anti-Bcl-2, anti-CHOP, anti-Bad and phosphorylated Bad (p-Bad), anti-Erk1/2 and phosphorylated Erk1/2 (p-Erk1/2), anti-Akt and phosphorylated Akt (p-Akt) antibody were all obtained from Cell Signaling Technology (Danvers, MA, USA). The anti-PERK and phosphorylated PERK (p-PERK) antibodies were obtained from Santa Cruz Biotechnology.
Anti tsg101
Manufactured by System Biosciences
Sourced in United States, United Kingdom
Anti-TSG101 is a laboratory reagent used to detect the presence of the TSG101 (Tumor Susceptibility Gene 101) protein. TSG101 is a key component of the endosomal sorting complex required for transport (ESCRT) machinery and plays a role in various cellular processes, including protein trafficking and cell division. Anti-TSG101 can be used in techniques such as Western blotting, immunoprecipitation, and immunocytochemistry to identify and quantify the levels of TSG101 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd63, by System Biosciences (38 mentions)
Anti cd9, by System Biosciences (11 mentions)
Anti cd81, by System Biosciences (10 mentions)
Anti hsp70, by System Biosciences (9 mentions)
Anti chop, by Cell Signaling Technology (3 mentions)
Anti calnexin, by Cell Signaling Technology (3 mentions)
Anti collagen 1, by Abcam (3 mentions)
Anti bcl 2, by Cell Signaling Technology (3 mentions)
Anti β catenin, by Cell Signaling Technology (3 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Anti pdgf bb, by Abcam (3 mentions)
Anti bfgf, by Cell Signaling Technology (3 mentions)
Phosphorylated erk1 2 p erk1 2, by Cell Signaling Technology (3 mentions)
Anti runx2, by Cell Signaling Technology (3 mentions)
Anti β tubulin, by Cell Signaling Technology (3 mentions)
Anti vegf, by Abcam (3 mentions)
Anti tgf β, by Cell Signaling Technology (3 mentions)
Ripa kit, by Beyotime (3 mentions)
Bovine serum albumin (bsa), by Beyotime (3 mentions)
Polyvinylidene difluoride membrane, by Beyotime (3 mentions)
7 protocols using anti tsg101
1
Comprehensive Protein Expression Analysis
2
Protein Analysis of iPSCs and sEVs
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The total proteins content in the iPSCs was extracted by a RIPA kit (Beyotime, Shanghai, China), and the protein samples of the sEVs were detected directly without extracted by a RIPA kit. The total amount of protein was detected by bicinchoninic acid method (BCA). All the samples were loaded in equal amount (15 μg), and then separated by a 10% SDS‐polyacrylamide gel, transferred to a polyvinylidene difluoride membrane (Beyotime, Shanghai, China). The membrane was blocked with 5% bovine serum albumin in TBST (Beyotime, Shanghai, China) and incubated overnight with primary antibodies. The blots were then washed with TBST, incubated with anti‐rabbit or anti‐mouse secondary antibodies (Cat. No. S0001 and S0002. Affinity Bioscience, OH, USA) and detected with the ECL‐2 reagent (Beyotime, Shanghai, China). The primary antibodies that were used in this study were anti‐HSP70, anti‐CD63, anti‐TSG101, anti‐calreticulin, and anti‐β‐actin which were all purchased from System Biosciences (SBI, Palo Alto, CA, USA) or Abcam (Cambridge, UK). Detailed information of antibodies was shown in the Table S1.
3
Exosomal Protein Isolation and Analysis
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Total proteins were isolated from exosomes using RIPA buffer (Sigma-Aldrich, St Louis, MO, USA) and the protein concentration was measured by Bradford assay (Beyotime, China). Exosomal proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) according to the manufacturer’s protocol. The membranes were probed using rabbit anti-CD63 (1:5000) and anti-TSG101 antibodies (1:3000) (System Biosciences) and the results were visualised using a Gel Doc XR+ system (Bio-Rad Laboratories, Hercules, CA, USA). Based on lncRNA sequence information and computational methods, the subcellular location of the H19 molecule was predicted using bioinformatics analysis tools (lncLocator: http: //www.csbio.sjtu.edu.cn/bioinf/lncLocator/index.html ).
4
EV Protein Expression Analysis
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For protein extraction, hUCB-MSC-derived EVs were lysed with RIPA buffer. Equal amounts of proteins from each group were separated by 10% sodium dodecyl sulfate (SDS) PAGE and transferred to a PVDF membrane. Anti-CD63 (System Biosciences, LLC, Palo Alto, CA, USA), anti-TSG101 (System Biosciences), anti-CANX (ABclonal, Woburn, MA, USA), and anti-GM130 (ABclonal) were used as primary antibodies. Horseradish peroxidase- (HRP-) conjugated IgG antibody (ABclonal) was used as the secondary antibody. Antibody-bound proteins were visualized using an enhanced chemiluminescent reagent.
5
Western Blot Analysis of Extracellular Vesicle Markers
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Protein samples were diluted 1:5 with protein loading buffer (6 ×; Transgen Biotech, Beijing, China) and heated at 95 °C for 5 min. Protein extracts were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, blotted onto polyvinylidene fluoride membranes, and blocked with 5% non-fat dried milk in TBST. The membranes were incubated with primary antibodies at 4°C overnight and with the horseradish peroxidase (HRP)-conjugated secondary antibodies at 37 °C for 2 hours. The primary antibodies (anti-CD9, anti-CD63, anti-TSG101, and anti-CD81) were obtained from SBI (System Biosciences; Palo Alto, CA, USA) and the secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Protein bands were visualized by enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA) and imaged by a FluorChem E gel documentation system (ProteinSimple, San Jose, CA).
6
Exosome Protein Marker Analysis by Western Blot
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Western blot analysis was performed as previously described, with the following antibodies at dilution 1:1000: anti-CD63, anti-CD9 and anti-TSG101 (SBI, Cat.NO.EXOAB-CD63A-1, EXOAB-CD9A-1 and EXOAB-TSG101-1), anti-ALIX (AbCam, Cat. No. ab117600), anti-C1q (CompTech, Cat. No. A200), anti-C1s (Quidel, Cat. No. A302). IRDye Donkey anti-rabbit or anti-goat was used as secondary antibodies (Rockland, Cat. No. 611-731-127) (LI-COR, Cat. No. 925-32213). Immuno-reactive bands were visualized by using the Odyssey digital imaging program.
7
Western Blot Analysis of Extracellular Vesicles
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Western blot samples were lysed in Pierce RIPA Buffer (Thermo Scientific, Waltham, USA). For protein measurement, BCA Protein Assay (Thermo Scientific, Waltham, USA) was used.
Primary antibodies targeting Syntenin (clone EPR8102, 1:1000 diluted) were purchased from Abcam (Cambridge, UK), anti‐CD63 antibodies (cat. no. EXOAB‐CD63A‐1, 1:2500 diluted) were from SBI (Palo Alto, California, USA), anti‐TSG101 (cat. no. 5701, 1:1000 diluted) from Sigma (Taufkirchen, Germany). Non‐EV proteins were stained using anti‐Calnexin (cat. no. 610523, 1:1000 diluted, BD Biosciences, San Jose, California, USA) and anti‐albumin antibodies (cat. no. Ab8940, 1:1000 diluted, Abcam, Berlin, Germany). Secondary antibodies (anti‐mouse IgG Peroxidase, cat. no. A0168 and anti‐rabbit IgG Peroxidase, cat. no. A0545) were purchased from Sigma‐Aldrich (St. Louis, Missouri, USA) and diluted 1:10,000. Hepatitis B core Antigen targeting antibody (anti‐HBcAg; cat. no. B0586, 1:1000 diluted) was purchased from Dako (Hamburg, Germany). Hepatitis B virus Surface Ad/Ay Polyclonal Antibody (cat. no. PA1‐73080, 1:10 diluted) was used for affinity‐based purification and purchased from Invitrogen (Carlsbad, California, USA). Recombinant HBV was produced as previously described and purified via heparin binding columns and cesium chloride gradient centrifugation (Burwitz et al.,2017 ).
Primary antibodies targeting Syntenin (clone EPR8102, 1:1000 diluted) were purchased from Abcam (Cambridge, UK), anti‐CD63 antibodies (cat. no. EXOAB‐CD63A‐1, 1:2500 diluted) were from SBI (Palo Alto, California, USA), anti‐TSG101 (cat. no. 5701, 1:1000 diluted) from Sigma (Taufkirchen, Germany). Non‐EV proteins were stained using anti‐Calnexin (cat. no. 610523, 1:1000 diluted, BD Biosciences, San Jose, California, USA) and anti‐albumin antibodies (cat. no. Ab8940, 1:1000 diluted, Abcam, Berlin, Germany). Secondary antibodies (anti‐mouse IgG Peroxidase, cat. no. A0168 and anti‐rabbit IgG Peroxidase, cat. no. A0545) were purchased from Sigma‐Aldrich (St. Louis, Missouri, USA) and diluted 1:10,000. Hepatitis B core Antigen targeting antibody (anti‐HBcAg; cat. no. B0586, 1:1000 diluted) was purchased from Dako (Hamburg, Germany). Hepatitis B virus Surface Ad/Ay Polyclonal Antibody (cat. no. PA1‐73080, 1:10 diluted) was used for affinity‐based purification and purchased from Invitrogen (Carlsbad, California, USA). Recombinant HBV was produced as previously described and purified via heparin binding columns and cesium chloride gradient centrifugation (Burwitz et al.,
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