Total protein was extracted from FLSs and BMSC exosomes using RIPA buffer (Biosharp, BL521A) containing protease inhibitors (Thermo, A32955) following the manufacturer’s instructions. Proteins were quantified using the BCA™ Protein Assay Kit (Biosharp, BL521A). A total of 30 μg of proteins were separated in 10–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The primary antibody solution was prepared in 5% blocking buffer. Primary antibodies including anti-BATF2 (16,592-1-AP, 1:1000, Proteintech), anti-STAT3 (ab68153, 1:1000, Abcam), anti-Alix (ab134045, 1:1000, Abcam), anti-CD9 (ab236630, 1:1000, Abcam), and anti-CD63 (ab134045, 1:2000, Abcam), anti-p-STAT3 (phospho Y705, ab267373, 1:1000, Abcam), anti-JAK2 (ab108596, 1:1000, Abcam), anti-p-JAK2 (phospho Y1007 + Y1008, ab32101, 1:2000, Abcam) and anti-GAPDH (ab8245, 1:5000, Abcam) were incubated with the membrane at 4°C overnight, followed by a brief wash and incubation with secondary antibody anti-mouse IgG (BA1051, 1:10,000, BOSTR) or anti-rabbit IgG (BA1054, 1:15,000, BOSTR) for 1 h at room temperature. The immuno-complexes were finally detected by ECL after washing by TBST and the bands were analyzed using the ImageJ software. The uncorrupted full size Western blot images were provided in supplementary materials S5.
Ab134045
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab134045 is a polyclonal antibody raised in rabbit against a synthetic peptide. This antibody is designed for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab125011, by Abcam (65 mentions)
Ab92726, by Abcam (57 mentions)
Ab109201, by Abcam (35 mentions)
Pvdf membrane, by Merck Group (22 mentions)
Ab236630, by Abcam (18 mentions)
Ab205718, by Abcam (16 mentions)
Ab79559, by Abcam (16 mentions)
Ab6721, by Abcam (15 mentions)
Ab133615, by Abcam (14 mentions)
Ab8245, by Abcam (12 mentions)
Ab8227, by Abcam (11 mentions)
Ab92573, by Abcam (11 mentions)
Ab263019, by Abcam (10 mentions)
Ab181602, by Abcam (10 mentions)
Ripa lysis buffer, by Beyotime (9 mentions)
Ab8226, by Abcam (9 mentions)
Ab32503, by Abcam (8 mentions)
Ab181606, by Abcam (8 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Ab22595, by Abcam (8 mentions)
191 protocols using ab134045
1
Protein Extraction and Western Blot Analysis
2
Exosome Isolation and Characterization
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Exosomes were precipitated by using an exosome precipitation solution (Exo-Quick; System Biosciences) following the manufacturer's instructions [19 (link)]. Ultrastructure and size distribution of the purified exosomes were analyzed by transmission electron microscopy (TEM) and NanoSight (Malvern), respectively. Protein markers, CD9 (Ab92726, Abcam, UK) and CD63 (Ab134045, Abcam, UK), were determined by immune blotting.
3
Exosomal Protein Extraction and Analysis
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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).
4
Exosome Identification via Western Blotting
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Exosome-specific markers, including positive (CD9, CD63, and TSG101) and negative markers (GRP94), were used to identify exosomes by western blotting. Total proteins (25 µg) in the extracted resuspension of exosomes were sequentially subjected to gel electrophoresis (10% SDS-PAGE), membrane transfer, blocking, incubation with primary antibodies specific for CD9, CD63, TSG101, and GRP94 (ab92726, ab134045, ab125011, ab238126, Abcam, Cambs, UK), incubation with the goat anti rabbit secondary antibodies, and enhanced chemiluminescence (ECL) to examine exosomal protein expression (22 (link), 23 (link)).
5
Protein Extraction and Western Blot Analysis
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Protein extraction and Western blot analysis were performed as described before.17 (link) Antibodies against specific proteins, including VEGF (Abcam, ab214424), PIAS3 (Cell Signaling Technology, 9042), p-STAT3 (Cell Signaling Technology, 9145), STAT3 (Cell Signaling Technology, 12640), CD63 (Abcam, ab134045) and TSG101 (Abcam, ab125011), were used.
6
Extracellular Vesicle Protein Quantification
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Extracellular vesicle proteins were quantified using the bicinchoninic acid (BCA) assay. Then, 25 μg of protein was put on 10% gradient SDS-PAGE gels and transferred onto polyvinylidene fluoride. And then the polyvinylidene fluoride was blocked with 5% skimmed milk and subsequently incubated overnight at 4°C with specific primary antibodies such as anti-CD63 antibody (abcam, ab134045), and anti-CD81 antibody (abcam, ab109201). After being washed four times, the polyvinylidene fluoride was incubated with a specific secondary antibody for 1 h, and proteins were detected using the enhanced chemiluminescence method with horseradish peroxidase kit (Thermo Fisher Scientific, Waltham, MA, United States) and visualized by a gel imaging system (AI600, GE Healthcare, United States).
7
Western Blot Analysis of Extracellular Vesicle Markers
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The whole extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) in an electrophoresis instrument (Bio-Rad, USA) and then transferred to a polyvinylidene difluoride (PVDF) membrane. Next, the membrane was washed in TBS Tween 20 and then incubated with primary antibodies against CD63 (1 : 1000, ab134045, Abcam), TSG101 (1 : 1000, ab125011, Abcam), Bax (1 : 1000, ab32503, Abcam), Bcl-2 (1 : 1000, ab32124, Abcam), cleaved-caspased3 (1 : 1000, ab32042, Abcam), cleaved-caspase9 (1 : 1000, ab2324, Abcam), MMP2 (1 : 1000, ab92536, Abcam), MMP9 (1 : 1000, ab76003, Abcam), and β-actin (1 : 1000, ab6276, Abcam). The membrane was washed for 3 times and incubated with the corresponding secondary antibody. The protein bands were observed with ECL chemiluminescence kit (Thermo Fisher Scientific, USA).
8
Quantitative Western Blot Analysis
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The total protein was extracted from tissues or cells with radioimmunoprecipitation assay lysis buffer (R0010, Solarbio), with the concentration determined by BCA Kit (20201ES76, YEASEN Biotech Co., Ltd., Shanghai). After separation by polyacrylamide gel electrophoresis, the protein was transferred to the PVDF membrane by wet transfer method. The membrane was sealed with 5% BSA at room temperature for 1 h, probed with the primary antibodies to CMTM7 (#PA5-103744, 1:1000, Thermo Fisher), EGFR (ab52894, 1:1000, Abcam), p-EGFR (ab40815, 1:1000, Abcam), AKT (ab8805, 1:500, Abcam), p-AKT (ab8933, 1:500, Abcam), VEGF (ab32152, 1:1000), CD63 (ab134045, 1:1000, Abcam), Hsp70 (ab181606, 1:1000, Abcam), TSG101 (ab125011, 1:2000, Abcam), and Calnexin (ab133615, 1:1000, Abcam) at 4 °C overnight. The next day, the membrane was re-probed with HRP labeled goat anti-rabbit IgG (ab205718, 1:10,000, Abcam) for 1 h at room temperature, developed by VILBER FUSION FX5 (VILBER LOURMAT, France). Image J 1.48u software (National Institutes of Health) was used for protein quantitative analysis, and the gray value of each protein was compared with the gray value of internal reference GAPDH.
9
Western Blot Analysis of Cellular Proteins
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Protein samples isolated from cultured cells using the RIPA lysis buffer (Cat# P0013C, Beyotime) were electrophoresed on 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking for 1 h with 5% skim milk, the membranes were incubated with specific primary antibodies at 4 °C overnight, followed by the secondary antibodies at 37 °C for 2 h. The immunoreactive signals were visualized by enhanced chemiluminescence reagent kit (Millipore). The primary antibodies used in this study: anti-PAK1 (1:1000, Cat#2602, Cell signaling, Boston, MA, USA), anti-CD63 (1:1000, ab134045, Abcam, Shanghai, China), anti-CD9 (1:2000, ab92726, Abcam), and anti-GAPDH (ab18602, 1: 5000, Abcam) [26 (link)].
10
Western Blot Analysis of Protein Markers
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Tissues and cells were lysed by RIPA lysis buffer (P0013b, Beyotime, China). Proteins were separated using SDS-PAGE and blotted onto PVDF membranes (Millipore, USA). The blots were then blocked with 5% non-fat milk and incubated with specific primary antibodies against CD9 (ab307085, Abcam, USA), CD63 (ab134045, Abcam, USA), CD81 (ab79559, Abcam, USA), TSG101 (ab125011, Abcam, USA), Calnexin (ab133615, Abcam, USA), Bcl-2 (ab32124, Abcam, USA), Bax (ab32503, Abcam, USA), cleaved Caspase-3 (ab2303, Abcam, USA), Caspase-3 (ab32351, Abcam, USA), GFAP (ab7260, Abcam, USA), NF200 (ab134306, Abcam, USA), TLR4 (ab13556, Abcam, USA), p65 (ab32536, Abcam, USA), IκB (9242, CST, USA), pIκB (9246, CST, USA), H3 (ab1791, Abcam, USA), β-actin (ab8226, Abcam, USA) overnight at 4 °C. Next day, the blots were probed with HRP-conjugated anti-rabbit (ab205718, Abcam, USA) or anti-mouse (ab205719, Abcam, USA) secondary antibodies at room temperature for 1 h. After reaction with ECL reagent (Millipore, USA), the protein bands were visualized by a gel image system.
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