Total proteins were exacted from cells and exosomes using lysis buffer (Beyotime). The protein concentration was analyzed using a BCA Protein Assay Kit (Beyotime). Proteins were separated using SDS‐PAGE and then transferred onto PVDF membranes (Roche Applied Science). The membranes were blocked with 2% BSA (Sigma‐Aldrich), followed by incubation with the indicated primary Abs overnight at 4℃. Membranes were then incubated with HRP‐conjugated goat anti‐mouse or goat anti‐rabbit IgG for 2 hours at room temperature, and the immunoblots were visualized using an enhanced chemiluminescence kit (GE Healthcare). The following primary Abs were used: anti‐heparanase (sc‐25825; Santa Cruz Biotechnology), anti‐CD63 (ab134045, 1:1000; Abcam), anti‐CD9 (ab92726, 1:1000; Abcam), anti‐CD81 (ab219209, 1:1000; Abcam), anti‐Flotillin‐1 (ab133497, 1:10 000; Abcam), and anti‐β‐actin (Abcam).
Ab133497
Manufactured by Abcam
Sourced in United Kingdom
Ab133497 is a recombinant monoclonal antibody. It is a laboratory reagent designed for research use.
Automatically generated - may contain errors
Lab products found in correlation
Ab1791, by Abcam (2 mentions)
Ab19903, by Abcam (2 mentions)
Ab52649, by Abcam (2 mentions)
Ab219209, by Abcam (1 mentions)
Ab134045, by Abcam (1 mentions)
Ab92726, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti β actin, by Abcam (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Ab6102, by Abcam (1 mentions)
Ab1318, by Abcam (1 mentions)
Western lighting chemiluminescence reagent, by PerkinElmer (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (1 mentions)
Na934 1ml, by GE Healthcare (1 mentions)
Shi exo m01, by Cosmo Bio (1 mentions)
Ma5 13548, by Thermo Fisher Scientific (1 mentions)
Na931 1ml, by GE Healthcare (1 mentions)
13 protocols using ab133497
1
Exosomal Protein Extraction and Analysis
2
Western Blot Analysis of EV Markers
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50 μL of EV samples from Sham and TAC EV was directly mixed with 5x SDS sample buffer, boiled at 95°C for 3 minutes and then separated on a SDS‐PAGE gel. The total loaded protein amount was calculated by the intensity of Coomassie blue stain. The membrane was blocked in 5% skim milk solution and incubated overnight with an antibody directed against hnRNA A2B1 (Ab6102, Abcam, 1:5000), CD63 (Ab1318, Abcam, 1:1000), Flotillin 1 (Ab133497, Abcam, 1:10 000), TSG101 (Ab83, Abcam, 1:1000), H3 (Ab1791, Abcam, 1:1000) and SERCA2a (custom antibody from 21st Century Biochemicals, 1:3000). The membrane was then incubated with a horseradish peroxidase‐conjugated secondary antibody (Sigma) and developed with Western Lighting chemiluminescence reagent (PerkinElmer).
3
Extracellular Vesicle Protein Analysis
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Purified EVs were lysed with 2× sodium dodecyl sulfate (SDS) sample buffer [100 mM Tris-HCl, pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol]. Total proteins were separated by SDS-PAGE, and then the following primary antibodies were used: anti-CD63 (mouse monoclonal, SHI-EXO-M02, 1:500, COSMO BIO Co. Ltd., Tokyo, Japan), anti-CD9 (mouse monoclonal, SHI-EXO-M01, 1:500, COSMO BIO Co. Ltd), anti-CD81 (mouse monoclonal, MA5-13548, 1:100, Thermo Fisher Scientific), anti-flotillin 1 (rabbit monoclonal, ab133497, 1:10,000, abcam). Subsequently, the following secondary antibodies were used: horseradish peroxidase (HRP)-conjugated anti-mouse IgG (NA931-1ML, 1:4,000, GE Healthcare UK Ltd, England), anti-rabbit IgG-HRP (NA934-1ML, 1:1,000, GE Healthcare UK Ltd). Protein bands were quantified by densitometry using Image Quant LAS 4000 (GE Healthcare UK Ltd, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England).
4
Protein Expression Quantification Protocol
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A previously described procedure was used24 with the following primary antibody (SERT: 1:1000, sc‐1458, Santa Cruz Biotechnology; Flotillin‐1:1:1000, ab133497, Abcam; β‐actin: 1:2000, A0760‐40, US Biological, Salem, Massachusetts) which were incubated with membranes overnight at 4°C. Target protein densitometry values were normalized to those of the housekeeping protein to semi‐quantitatively determine protein levels.
5
Comprehensive Western Blotting Protocol
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Western blotting was performed as previous reported70 (link). Briefly, equal molarity of protein extracts was loaded and separated in an SDS–PAGE, and transferred to a PVDF membrane. Immunoblot analysis was performed with overnight incubation of anti-vimentin (1:1000; 5741, Cell Signaling), anti-claudin1 (1:1000; 4933, Cell Signaling), anti-Snail (1:1000; 3879, Cell Signaling), anti-Twist1 (1:1000; 711565, Invitrogen), anti-ZEB1 (1:1000; 3396, Cell Signaling), anti-ZEB2 (1:1000; 97885, Cell Signaling), anti-ATP1A1 (1:10000; 14418-1-AP, Proteintech), anti-ATP1A2 (1:1000; 16836-1-AP, Proteintech), anti-ATP1A3 (1:1000; 28030-1-AP, Proteintech), anti-EMC1 (1:1000; GTX119884, Genetex), anti-ITGB1 (1:1000; 12594-1-AP, Proteintech), anti-αSMA (1:1000; ab7817, abcam), anti-collagen1 (1:1000; ab34710, abcam), anti-Flag (1:1000; F3165, Sigma), anti-p-p65 (S536) (1:1000; 3033, Cell Signaling), and anti-p65 (1:1000; 3034, Cell Signaling). anti-GAPDH (1:10000; GTX627408, Genetex) was used as loading controls for total cell lysates or cytosolic fraction. anti-flotillin1 (1:1000; ab133497, abcam) was used as loading control for membrane fraction.
6
Extracellular Vesicle Protein Profiling
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Concentrated conditioned media samples were subjected to Western blot to visualize relevant protein markers. First, IP was performed on samples as described above. A Bradford assay was conducted to determine protein concentrations, and 5 μg of each sample was run on an AnykD Mini-PROTEAN TGX Pre-Cast Gel (Bio-Rad) at 135 V for 50 minutes. Protein was transferred to a PVDF membrane using a Bio-Rad Trans-Blot Turbo. Membranes were blocked in 5% skim milk for 1 hour at room temperature and incubated with the following primary antibodies overnight at 4°C: anti-HSP90 (37-9400; mouse monoclonal; Thermo Fisher Scientific), anti-CD63 (25682-1-AP; rabbit polyclonal; Thermo Fisher Scientific), anti-flotillin1 (ab133497; rabbit monoclonal; Abcam), anti-syntenin1 (ab19903; rabbit polyclonal; Abcam), and anti–histone H3 (ab1791; rabbit polyclonal; Abcam); these markers were selected based on the Minimal Information for Studies of Extracellular Vesicles 2018 guidelines (19 (link)) and a recent comprehensive overview of EV protein markers (53 (link)). Following primary antibody incubation, membranes were washed in 1× Tris-buffered saline with Tween 20 (TBS-T) and incubated with the corresponding IRDye 800CW goat anti-mouse or anti-rabbit secondary antibody (LI-COR Biosciences) for 1 hour at room temperature. Membranes were subsequently washed in 1× TBS-T and imaged on a LI-COR Odyssey CLx.
7
Isolation and Characterization of NSC-derived Exosomes
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The culture supernatants of NSCs were collected for isolating exosomes, referring to previous protocols (Rong et al., 2019; Li et al., 2020a; Pan et al., 2020). Briefly, the growth medium was collected, and dead cells, cellular debris, and larger microvesicles were removed by sequential centrifugation (300 × g for 10 minutes, followed by 1000 × g for 10 minutes and 20,000 × g for 30 minutes at 4°C) and filtration through a 0.22-µm filter (Millipore, Burlington, MA, USA). The total concentration of exosomes was determined using a bicinchoninic acid (BCA) kit (Sigma). NSC-ex morphology was assessed using a transmission electron microscope (Philips CM 120; Philips, Amsterdam, Netherlands). The size distribution of the exosomes was analyzed by nanoparticle tracking analysis, as we described previously (Li et al., 2020a). Furthermore, the exosome-specific markers CD63 (1:1000, Abcam, Cambridge, UK, Cat# ab217345, RRID: AB_2754982), Alix (1:1500, Abcam, Cat# ab117600), Flotillin1 (1:10,000, Abcam, Cat# ab133497, RRID: AB_11156367), and TSG101 (1:2000, Abcam, Cat# ab125011, RRID: AB_10974262) were detected by western blot analysis, and Calnexin (1:2000, Abcam, Cat# ab133615, RRID: AB_2864299) was detected as a negative control.
8
Immunofluorescence Staining of Paraffin-Embedded Tissues
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Patient specimens and mouse cell line-derived xenografts were formalin-fixed and paraffin-embedded. Embedded tissues were sectioned at a thickness of 5 μm for hematoxylin and eosin (H&E) or immunofluorescence (IF) analysis. For immunofluorescence staining, after dewaxing and hydration, the sections were subjected to antigen repair to reveal epitopes by using sodium citrate antigen retrieval solution (C1032, Solarbio). Then, the sections were incubated with 5% bovine serum albumin (AP36L014, Life-iLab) for 1 h and incubated overnight at 4 °C with primary antibodies against 4-HNE (ab46544, goat, 1:100, Abcam) and FLOT1 (ab133497, rabbit, 1:100, Abcam). The next day, sections were washed and incubated with the appropriate secondary antibody DyLight 488 Conjugate anti-goat IgG (E032231, donkey, 1:200, EarthOx) or DyLight 594 Conjugate anti-rabbit IgG (E032421, donkey, 1:200, EarthOx) for 90 min at room temperature. Then, anti-fade mounting medium with DAPI (S2100, Solarbio) was mounted. Fluorescence images of sections were acquired by the Vectra® Polaris™ Automated Quantitative Pathology Imaging System.
9
Immunoprecipitation and Protein Analysis
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Cells were lysed with ice-cold immunoprecipitation buffer [10 mM tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 60 mM n-octyl-β-d -glucopyranoside (Sigma-Aldrich)] containing protease and phosphatase inhibitors. Protein concentrations from cells were determined using Quick Start Bradford Protein Assay reagent (Bio-Rad). Protein (3 to 4 mg/ml) in immunoprecipitation buffer was incubated with control rabbit immunoglobulin G (10 μg/ml; Thermo Fisher Scientific) or monoclonal antibodies to flotillin-1 (ab133497, Abcam) or MMP14 (ab51074, Abcam) overnight on a vertical rotator at 4°C. After, protein G agarose beads (100 μl/ml; Roche) were added and incubated for a further hour. Bound protein complexes were spun down, washed twice with immunoprecipitation buffer and once with 1× TBS, and eluted by heating in 4× Laemmli sample buffer (Bio-Rad) containing reducing agent (Bio-Rad) for 10 min at 95°C for analysis by Western blotting.
10
Profiling EV Protein Markers
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MSC-EVs lysates were prepared using 30X ethanol precipitation into RIPA lysis buffer. Lysates were volume loaded and separated on a 12% Bolt Bis-Tris Gel and probed using Abcam antibodies against flotillin-1 (ab133497 at 1:10,000 dilution), Annexin-2 (ab41803 at 1:1000 dilution), Syntenin-1 (ab19903 at 1:1000 dilution), MHC-I (ab110645 at 1:1000 dilution), MHC-II (157210 at 1:10000 dilution), and Calreticulin (ab92516 at 1:1000 dilution).
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