Extracellular vesicle protein was prepared by re-suspending EV pellets in RIPA buffer supplemented with protease inhibitor (Roche). Twenty micrograms of protein from each sample was separated on a 10% SDS-PAGE gel in parallel with a protein marker (Thermo). Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, and blocked with 5% skim milk dissolved in Tween/Tris buffered saline (TTBS) for 1 h at room temperature. Membranes were then incubated with primary antibodies against TSG101 (ProteinTech) or CD9 (ProteinTech) at a dilution of 1:5000 with 3% skim milk diluted in TTBS for 1 h at room temperature. After washing 6 times with TTBS, membranes were incubated with horseradish peroxidase conjugated goat anti-rabbit IgG secondary antibodies (Santa Cruz Biotechnology) at a dilution of 1:10,000 in 3% skim milk dissolved in TTBS for 1 h at room temperature. After washing 6 times with TTBS, membranes were covered with ECL western blot substrate solution and visualized by exposing to film and developing in a film processor.
Tsg101
Manufactured by Proteintech
Sourced in United States, China
TSG101 is a protein that plays a role in the formation of endosomal sorting complexes required for transport (ESCRT) machinery. It is involved in the budding of vesicles from the plasma membrane and in the sorting of ubiquitinated proteins into multivesicular bodies.
Automatically generated - may contain errors
Lab products found in correlation
Calnexin, by Proteintech (13 mentions)
Pvdf membrane, by Merck Group (11 mentions)
Gapdh, by Proteintech (9 mentions)
Gapdh, by Cell Signaling Technology (7 mentions)
Hsp70, by Proteintech (7 mentions)
β actin, by Proteintech (7 mentions)
Bcl 2, by Proteintech (6 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Bcl2 associated x (bax), by Proteintech (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (3 mentions)
Ripa buffer, by Santa Cruz Biotechnology (3 mentions)
Calnexin, by Abcam (3 mentions)
Gapdh, by Abcam (3 mentions)
Il 1β, by Proteintech (3 mentions)
Tnf α, by Proteintech (3 mentions)
P akt, by Proteintech (3 mentions)
Beclin 1, by Proteintech (3 mentions)
Vamp3, by Proteintech (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
74 protocols using tsg101
1
Western Blot Analysis of Extracellular Vesicles
2
Protein Extraction and Western Blot Analysis
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Protein extraction and western blot (WB) analysis were performed as previously described [14 (link)]. Briefly, cells were washed with PBS and lysed with lysis buffer on ice for 20 min. The total cell protein concentration was detected using the BCA Protein Assay Kit. The total protein (20 μg) was separated using SDS-PAGE (Invitrogen) and transferred to a PVDF membrane (Roche). The membrane was blocked with 5% bovine serum albumin (0.1%) in TBS-Tween and incubated against the required antibody.
The primary antibodies Bax (5023, Cell Signaling Technology, USA), Bcl2 (ab196495, Abcam, USA), cleaved caspase-3 (29034, Signalway Antibody, USA), BTG2 (A9848, ABclonal), GAPDH (5174, Cell Signaling Technology), TSG101 (14497, Proteintech, United States), CD63 (25682, Proteintech), CD81 (66866, Proteintech), and horseradish peroxidase-conjugated secondary antibody (Santa Cruz) were used. Bands were visualized using enhanced chemiluminescence reagents and analyzed using a gel documentation system (Bio-Rad Gel Doc1000 and Multi-Analyst version 1.1).
The primary antibodies Bax (5023, Cell Signaling Technology, USA), Bcl2 (ab196495, Abcam, USA), cleaved caspase-3 (29034, Signalway Antibody, USA), BTG2 (A9848, ABclonal), GAPDH (5174, Cell Signaling Technology), TSG101 (14497, Proteintech, United States), CD63 (25682, Proteintech), CD81 (66866, Proteintech), and horseradish peroxidase-conjugated secondary antibody (Santa Cruz) were used. Bands were visualized using enhanced chemiluminescence reagents and analyzed using a gel documentation system (Bio-Rad Gel Doc1000 and Multi-Analyst version 1.1).
3
Western Blot Analysis of Macrophage and Exosome Proteins
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Total proteins were isolated from fresh macrophages cells (5.5 × 106) or frozen exosomes (100 μg) were lysed using RIPA peptide lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Proteins (30 μg) were loaded on 10% SDS-PAGE gels, electrophoresed, and then transferred to PVDF membranes (MilliporeSigma, Burlington, MA, United States). Subsequently, the membranes were incubated with primary antibodies against CD81 (at 1:2,000 dilution, ProteinTech Group, Inc., Chicago, United States), HSP70 (at 1:3,400 dilution, Biolegend, Inc., San Diego, CA, United States), LBP (at 1:2,400 dilution, ProteinTech Group, Inc.), CD36 (at 1:2,000 dilution, Biolegend, Inc.), MHC-I (at 1:3,000 dilution, Biolegend, Inc.) and TSG101 (at 1:2,000 dilution, ProteinTech Group, Inc.) at 4°C overnight. Then, PVDF membranes were washed three times with TBS-T under shaking. Finally, the membranes were incubated with secondary antibody solution (mouse anti-rabbit IgG-HRP at a 1:10,000) for 60 min at room temperature with gentle shaking, and washed with TBS-T under shaking three times again. Protein bands were imaged and analyzed using the Chemiluminescent Substrate System (Thermo Fisher Scientific, Inc., Waltham, MA, United States).
4
Exosomal Protein Profiling by Western Blot
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Cell or exosome lysates were diluted at a 1:5 ratio with loading buffer (6×) and heated at 95°C for 6 minutes. Protein extracts were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). Next, the membranes were blocked in 5% nonfat milk for 2 hours. Then, the membranes were incubated overnight with primary antibodies specific to CD9 (1:10,000, Abcam, MA), CD63 (1:5,000, Abcam), TSG101 (1:1,000, Proteintech Group, IL), GM130 (1:5,000, Proteintech Group), RUNX2 (1:3,000, Cell Signaling Technology, Beverly, MA), COL1 (1:6,000, Abcam), VEGFA (1:1,000, Abcam) and GAPDH (1:4,500, Cell Signaling Technology). Next, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibodies for 1 hour at room temperature. The immunoreactive bands were detected using a FluorChem WB imaging system (ProteinSimple, San Jose, CA).
5
Protein Extraction and Western Blot Analysis
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For protein extraction, cells were washed and suspended with RIPA buffer. The protein was obtained by centrifugation with 12,000 rpm for 30 min at 4°C. Protein was separated by 10% SDS-PAGE. The bands were transferred onto nitrocellulose membrane (PALL, United States), then blocked with 5% skim milk and incubated with primary conjugated antibodies (CD63: Proteintech, Cat No. 25682-1-AP; TSG101: Proteintech, Cat No. 67381-1-Ig; Bax: Proteintech, Cat No. 50599-2-Ig; Bcl-2: Proteintech, Cat No. 12789-1-AP; Cleaved Caspase3: CST, Cat No. 9661; Cleaved Caspase9: CST, Cat No. 7237; MCL1: Proteintech, Cat No. 16225-1-AP; β-actin: Proteintech, Cat no. 66009-1-Ig). Then the nitrocellulose membrane was incubated with the secondary conjugated antibody (HRP-conjugated Affinipure Goat Anti-Rabbit: Proteintech, Cat no. SA00001-2; HRP-conjugated Affinipure Goat Anti-Mouse IgG: Proteintech, Cat no. SA00001-1) for 4 h at room temperature and washed with TBST. The target protein bands were imaged by ECL (Advansta, United States), analyzed, and quantified by Bio-Rad ChemiDoc™ MP system (Bio-Rad, United States).
6
Isolation and Characterization of M2 Macrophage-Derived Exosomes
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RAW264.7 cells were induced to an M1/M2 type for 24 h and cultured in an exosome-depleted FBS-containing complete medium. The supernatant was collected, and exosomes were obtained through ultracentrifugation [35 (link)]. Briefly, the supernatant was subjected to a series of differential centrifugation steps (300×g for 10 min, 2000×g for 10 min, and 10,000×g for 30 min) to remove intact cells and cell debris. Subsequently, the supernatant was centrifuged at 100,000×g at 4 °C for 70 min to isolate the proteins-containing exosomes. The exosomes were then purified by washing them with PBS and subjected to an additional centrifugation step at 100,000×g for 70 min.
The protein content of the exosomes was measured with a bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai, China) referring to the manufacturer’s instructions. CD9, CD63, CD81, TSG101 and Calnexin (Proteintech, Chicago, USA), which were exosomal marker proteins, were assessed by western blotting as previously described [51 (link)]. M2 macrophage-derived exosomes were scanned using a TEM (JEM1400, Tokyo, Japan). The size and concentration of exosomes were analyzed through NTA (Nanosight NS300, Malvern, UK).
The protein content of the exosomes was measured with a bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai, China) referring to the manufacturer’s instructions. CD9, CD63, CD81, TSG101 and Calnexin (Proteintech, Chicago, USA), which were exosomal marker proteins, were assessed by western blotting as previously described [51 (link)]. M2 macrophage-derived exosomes were scanned using a TEM (JEM1400, Tokyo, Japan). The size and concentration of exosomes were analyzed through NTA (Nanosight NS300, Malvern, UK).
7
Protein Extraction and Western Blot Analysis
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Both types of cells were lysed on ice with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, USA) supplemented with 10% protease inhibitor cocktail (Roche, Mannheim, Germany) to isolate total protein and then measured by a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA) in accordance with the manufacturer’s instructions. Protein samples supplemented with loading buffer in equal proportions were electrophoresed on NuPAGE™ Bis-Tris Protein Gels (Invitrogen, Waltham, MA, USA) and then transferred onto polyvinylidene fluoride (PVDF) membranes. After incubation with 5% skim milk (5% w/v) for 2 h at room temperature, the membranes were co-incubated overnight at 4 °C with the following primary antibodies specific for GAPDH (1:1000; Cell Signaling Technology, USA), Runx2 (1:1000; Cell Signaling Technology, Danvers, MA, USA), Osx (1:1000; Abcam, Cambridge, UK), Ocn (1:2000; Abcam, UK), ELK4 (1:1000; Proteintech, Rosemont, IL, USA), GM130 (1:1000; Proteintech, USA), CD9 (1:1000; Proteintech, USA), and TSG101 (1:1000; Proteintech, USA). Next, the peroxidase-conjugated secondary antibody (1:5000; ZS-GB-BIO, Beijing, China) was utilized to treat the PVDF membranes for 2 h at room temperature. The ECL kit (Thermo Fisher Scientific, USA) was employed to visualize the signals. Densitometry analysis was conducted with ImageJ software.
8
Comprehensive Extracellular Vesicle Protein Analysis
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Purified sEV samples were incubated on ice in 10X RIPA buffer supplemented with protease inhibitor cocktail (Cell Signaling Technology, #9806) and proteosome inhibitor MG-132 (Sigma, #M7449). Either 4X reducing sample buffer or 4X LDS non-reducing sample buffer (Thermo Scientific, #84,788) was added to the sEVs before heating at 95°C for five minutes. sEV samples were electrophoresed on SDS-polyacrylamide gels and subsequently transferred to a methanol-activated Immobilon-P Transfer Membrane (#IPVH00010 Immobilon). After blocking at room temperature for one hour with blocking buffer (1X TBS pH 7.6 containing 0.05% Tween-20 and 5% fat-free dry milk), membranes were incubated at 4°C with rocking overnight using primary antibodies for the following proteins: ALIX (1/1,000, Proteintech #67,715-1-Ig), β-Actin (1/10,000, Santa Cruz Biotechnology, #sc-47778), CD-9 (1/1,000, Proteintech #20,597-1-AP), Flotillin 1 (1/1,000, Proteintech #15,571-1-AP), GAPDH (1/2,000, Cell Signaling Technology #5174), Hif-1α (1/1,000, Novus Biologicals #NB100-449), placental alkaline phosphatase (1/1,000, Boster Biological Technology #A01718), TSG101 (1/1,000, Proteintech #28,283-1-AP). Following incubation, membranes were subsequently washed and incubated with secondary antibody at 1/10,000 for 1 h at room temperature and visualized via West Pico SuperSignal chemiluminescence.
9
Quantification and Protein Analysis
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Bradford assay (5000202; BioRad, Hercules, CA, USA) was carried out to quantify cell lysates. Western blotting was performed using antibodies including HIF-1a (36169; Cell Signaling Technology), antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5174; Cell Signaling Technology), TSG101 (14497, Proteintech, United States), CD63 (25682, Proteintech) and CD81 (66866, Proteintech).
10
Western Blot Analysis of Viral and Cellular Proteins
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Western blot assay was carried out as follow: cells were lysed with RIPA buffer (Santa Cruz, USA) and cleared lysate was collected by centrifugation for protein separation on 10% SDS-polyacrylamide gel. The proteins were transferred onto PVDF membranes (Millipore) and detected with respective antibodies at 4°C overnight, followed by incubation with either IRDye Fluor 680-labeled IgG or IRDye Fluor 800-labeled IgG secondary antibody (Li-Cor Bioscience). The images were scanned and quantified by densitometric analysis by Li-COR Odyssey Infrared Imager. Primary antibodies against VP1 (Millipore), CD81 (Cell Signaling Technology), IRAK1 (Santa Cruz), CD63 (Abcam), ISG15 (Abcam) and Rab27a, TRAF6, STAT1, TSG101, BST-2/Tetherin, and GAPDH (all from Proteintech), VP2, 3AB, 3C and 3D (all from Genetex) were used.
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