Protein extracts from cells were obtained as previously described [61 (link)]. Briefly, proteins samples were extracted by homogenization using a TissueLyser II (QIAGEN, Tokio, Japan) in cold RIPA buffer (200 mM Tris/HCl (pH 7.4), 130 mM NaCl, 10% (v/v) glycerol, 0.1% (v/v) SDS, 1% (v/v) Triton X-100, 10 mM MgCl2) with anti-proteases and anti-phosphatases (Sigma-Aldrich; St Louis, MO, USA). Twenty-five μg cell lysates and the VEs obtained from the same amount of cells per plate from at least three independent experiments were separated in 10% SDS-PAGE gels and electroblotted onto nitrocellulose membranes as previously described [13 (link)]. Primary anti-PAkt, and anti-Akt were purchased from Cell Signaling Technology (MA, USA); anti- Alix, anti-Ceruloplasmin, anti-TSG101, anti-CD81, anti-Syntenin, and anti-Mimecan from Sta. Cruz Biotechnology (CA, USA); anti-TGFBeta Ig-h3 (NMP2-67186; dilution 1:500) from Novus Biologicals (NovusBio, CO, USA) and anti-Perilipin-1 from Abcam. Data were expressed as percentages corrected towards GADPH (arbitrary units) in Western blots with mean ± SEM. Data analyses were conducted using GraphPad Prism 6 software.
Anti cd81
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-CD81 is a lab equipment product from Cell Signaling Technology. It is an antibody that specifically binds to the CD81 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti alix, by Cell Signaling Technology (2 mentions)
Anti tsg101, by Cell Signaling Technology (2 mentions)
Anti cd9, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti cd63, by Abcam (2 mentions)
Anti cd9, by Abcam (2 mentions)
Anti cd63, by Cell Signaling Technology (2 mentions)
Anti tsg101, by Abcam (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Anti akt and anti pakt, by Cell Signaling Technology (1 mentions)
Anti perilipin 1, by Abcam (1 mentions)
Anti proteases and anti phosphatases, by Merck Group (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Bolt 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Mini hd9 western blot imaging system, by Cleaver Scientific (1 mentions)
Anti tsg101, by Proteintech (1 mentions)
10 protocols using anti cd81
1
Protein Extraction and Western Blot Analysis
2
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).
3
Western Blot Analysis of Exosomal Proteins
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Cells were lysed using a RIPA buffer, including a protease inhibitor cocktail (Thermo Scientific, USA). The proteins were separated by SDS-PAGE gels and transferred to PVDF membranes (Millipore, USA). After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies at 4°C overnight. The HRP-conjugated secondary antibodies were used to incubate the membranes for 2h at room temperature. The membranes were washed and incubated for 1h at room temperature with HRP-conjugated secondary antibodies. Proteins were detected using a Bio-Rad ChemiDoc XRS+System. Bio-Rad Image Lab software was used for densitometric analysis. The following primary antibodies were purchased: anti-CD81 (1:2000; Cell Signaling, USA), anti-TSG101 (1:1000; Proteintech, USA), anti-CD9 (1:1000; Abcam, USA), anti-CD63 (1:1000; Abcam, USA), anti-PTEN (1:1000; Cell Signaling, USA), anti-PI3K p85 (1:1000, Abacm, USA), the anti-phospho-PI3K p85 (1:1000, Cell Signaling, USA), anti-p-AKT (phosphor S473) (1:1000; Abcam, USA), anti-AKT (1:1000; Abcam, USA), anti-GAPDH (1:5000; Santa Cruz, CA).
4
Immunogold Labeling of Extracellular Vesicles
5
Western Blot Analysis of Exosome Markers
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The experimental procedure was performed as described in the previous study [32 (link)]. The total protein concentration of cells and tissues was measured using the BCA protein quantification method (Beyotime, China). Equal amounts of proteins were resolved by electrophoresis on 10% SDS gel and then transferred onto nitrocellulose membranes. Following blocked by incubation in 5% nonfat milk, membranes were probed with specific anti-CD9 (Cell Signaling Technology, 1:1000), anti- CD63 (Cell Signaling Technology, 1:1000), anti- CD81 (Cell Signaling Technology, 1:1000), anti- FGL1 (Abcam, 1:1500), anti-p-p65 (Abcam, 1:1500), anti-p65 (Abcam, 1:1000), anti-IκBα (Abcam, 1:1000), anti-p-IκBα (Abcam, 1:1500), anti-Bcl-2 (Abcam, 1:1500), anti-Bax (Abcam, 1:1500) and anti-cleaved-caspase 3 (Abcam, 1:1000) overnight at 4°C. After washing with Tris buffer saline (TBS) containing 0.24% Tween-20, membranes were then incubated for 60 min with horseradish peroxidase-conjugated secondary antibody. Then, an enhanced chemiluminescence system was used for visualization of protein signals, and the density of each protein band was analyzed using Image-Pro Plus6.0 (Media Cybernetics, Silver Spring, USA).
6
Comprehensive Protein Expression Analysis
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Cells and exosomes were lysed with RIPA lysis buffer containing protease and phosphatase inhibitors. Nuclear proteins were extracted with a kit (CWBIO, Beijing, China). Protein samples were separated by SDS-PAGE, and then transferred to PVDF membranes (Millipore, Burlington, MA, USA). After blocking for 1 h, the PVDF membranes were incubated with primary antibodies overnight at 4℃. The primary antibodies used were anti-CD81 (1：1,000; Cell Signaling Technology, Danvers, MA, USA), anti-CD63 (1：1,000; Cell Signaling Technology), anti-TSG-101 (1：1,000; Cell Signaling Technology), anti-calnexin (1：1,000; Cell Signaling Technology)，anti-Galectin-3 (1：1,500; Abcam, Cambridge, UK), anti-α-SMA (1：1,000; Bioworld Technology, Bloomington, MN, USA), anti-Collagen Ⅰ (1：1,000; Bioworld Technology), anti-Pe-riostin (1：1,000; Proteintech, Wuhan, China), anti-IL-1β (1：1,000; Bioworld Technology), anti-TNF-α (1：1,000; Bioworld Technology), anti-TGF-β (1：1,000; Abcam), anti-β-catenin (1：1,000; Cell Signaling Technology), anti-GAPDH (1：3,000; CWBIO, Beijing, China), and anti-Histone (1：1,000; Cell Signaling Technology). The next day, membranes were incubated at 37℃ for 1 h with hor-seradish peroxidase-linked goat anti-rabbit or anti-mouse antibodies (1：3,000; ABM, Richmond, Canada).
7
Flow Cytometric Analysis of CD81 Expression
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Dispersed islets or cultured cells were sorted and analyzed using FACS-Aria III (BD Bioscience). TrypLE Express Enzyme (1X) (Thermo Fisher Scientific) was used to detach Min6 from the dish or dissociate islets into single cells. Staining was performed using FACS buffer (PBS, 2% FBS and 2 mM EDTA). Cells were incubated with rabbit monoclonal anti-PE-CD81 (#93765, Cell Signaling, 1:50) on ice for 30 min. Alternatively, cells were stained for 30 min on ice with primary antibody (rabbit monoclonal anti-CD81, #10037, Cell Signaling, 1:50) and subsequently for 20 min on ice and in the dark with donkey anti-rabbit IgG Alexa Fluor 647 (A31573, Invitrogen, 1:500). Post-processing analysis was performed using FlowJo software.
8
Microglial Activation and Polarization
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The microglial activator lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies use for western blotting in our study included anti-β-actin (Abcam, Cambridge, UK), anti-TSG101 (Abcam), anti-CD9 (Abcam), anti-CD63 (Abcam), anti-CD81 (Cell Signaling Technology, Danvers, MA, USA), anti-iNOS (Abcam), anti-Arg1 (CST), anti-TLR4 (Abcam), anti-p-P65 (CST), anti-MyD88 (CST), anti-p-PI3K (CST), anti-PI3K (CST), anti-p-AKT (CST), and anti-AKT (CST). The antibodies used for immunofluorescence were anti-iNOS (Abcam), anti-Arg1 (CST), anti-Iba1 (Servicebio, Wuhan, China), anti-NF200 (Abcam), and anti-GFAP (Abcam). The secondary antibody was a cyanine 3- or FITC-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA). The TNF-α, IL-1β, IL-6, TGF-β, IL-4, and IL-10 ELISA kits were obtained from R&D Systems.
9
Western Blotting of Exosomal Markers
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Western blotting was performed as described previously (Hosoi et al., 2014 (link)) albeit with some modifications. Briefly, the cells were washed and lysed for 20 min with RIPA buffer containing 25 mM Tris HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% sodium deoxycholate, 1 mM Na3VO4, 10 mM NaF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The lysates were incubated on ice for 20 min. Then, the samples were sonicated for 4 min and suspended for 30 min at 4°C. After additional sonication for 4 min, the samples were centrifuged at 14,000 rpm for 5 min at 4°C, and the supernatants were collected. The samples were then boiled in SDS sample buffer for 3 min, fractionated with SDS-PAGE, and transferred to nitrocellulose membranes. These membranes were then incubated with anti-CD63 (Novus; 1:1000), anti-calnexin (Enzo; 1:1000), anti-CD81 (Cell signaling; 1:1000), and anti-IRE1α (Cell signaling; 1:1000) antibodies, followed by an anti-horseradish peroxidase-linked antibody. Peroxidase binding was detected using chemiluminescence with an enhanced chemiluminescence system.
10
Western Blotting of Protein Targets
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Proteins levels were analyzed by Western blotting using a standard protocol59 (link). Antibodies used in this study were: anti-Rab7 (9367S, 1:1000), anti-Alix (2171S, 1:1000), anti-CD81 (10037S, 56039S, 1:1000), anti-Tom20 (42406S, 1:1000), anti-Rab4 (2167S, 1:1000), anti-Rab5 (3547S, 1:1000), anti-Rab9 (5118S, 1:1000), anti-Rab11 (5589S, 1:1000), anti-LC3A/B (4108S, 1:1000), anti-Rab27 (69295S, 1:1000), anti-Arl8b (56085 S, 1:1000), anti-Atg5 (12994S, 1:1000), anti-Atg7 (2631S, 1:1000) from Cell Signaling Technology; anti-SQSTM1/p62 (ab56416, 1:1000), anti-Tsg101 (ab83, 1:1000), anti-Calreticulin (ab2907, 1:1000) from Abcam; anti-Tim23 (11123-1-AP, 1:1000) from Proteintech, anti-CD63 (PA5-92370, 1:500) from Invitrogen; anti-dendra2 (TA150090, 1:1000) from OriGene; anti-GAPDH (GTX627408, 1:2000) from GeneTex; Anti-Ubiquitin (P4D1) (sc-8017, 1:1000) from Santa Cruz, MTCO1 (459600, 1:1000) from Thermo Fisher Scientific, and MnSOD (06-984, 1:1000) from Millipore. Goat anti-mouse HRP (31430; 1:5000) and Goat-anti-rabbit HRP (31460, 1:5000) secondary antibodies from Thermo Fisher Scientific.
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