Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and antibiotic-antimycotic solution were obtained from Gibco BRL (Gaithersburg, MD, USA). Recombinant human TGF-β1 was obtained from R&D Systems (Minneapolis, MN, USA). Eupatilin and p38 inhibitor (SB203580) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-FN, anti-Col I, anti-Col III, and anti- αSMA were purchased from Abcam (Cambridge, MA, USA). Anti-phosphorylated-Smad2/3 (anti-p-Smad2/3), anti-Smad2/3, anti-phosphrylated-p38 (anti-p-p38), anti-p38, anti-glyceraldehyde-3-phosphate dehydrogenase, and the secondary antibody (anti-rabbit IgG) were obtained from Cell Signaling Technology (Danvers, MA, USA). Detailed information on the antibodies is provided in S1 Table .
Anti col 3
Manufactured by Abcam
Sourced in United States
Anti-Col III is a laboratory reagent that can be used to detect and quantify type III collagen in biological samples. It is a primary antibody that specifically binds to type III collagen, a component of the extracellular matrix in various tissues.
Automatically generated - may contain errors
Lab products found in correlation
Anti col1, by Abcam (7 mentions)
Anti α sma, by Abcam (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Anti sirt1, by Abcam (2 mentions)
Anti gapdh, by Proteintech (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Recombinant human tgf β1, by R&D Systems (1 mentions)
Anti fn, by Abcam (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase, by Cell Signaling Technology (1 mentions)
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
α sma, by Abcam (1 mentions)
Fam sirna, by GenePharma (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Sdf 1, by Thermo Fisher Scientific (1 mentions)
Anti tgf β, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Branched pei, by Polysciences (1 mentions)
Scrambled sirna siscr, by GenePharma (1 mentions)
10 protocols using anti col 3
1
Eupatilin Inhibits Fibrosis in Transforming Growth Factor-β1-Stimulated Lung Epithelial Cells
2
Cyclam-Branched PEI Nanoparticles for TGFβ siRNA Delivery
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Cyclam was bought from Vesina Industrial company (Tianjin, China). Branched PEI (MW = 10 kDa) was purchased from Polysciences (Warrington, PA, USA). AMD3100 was obtained from Biochempartner (Shanghai, China). TGFβ siRNA (5-GCAACAACGCCAUCUAUGATT-3), FAM siRNA (5-UUCUCCGAACGUGUCACGUTT-3), scrambled siRNA (siScr) (5-UUCUCCGAACGUGUCACGUTT-3), siRNA targeting luciferase (5-GGACGAGGACGA GCACUUCUU-3) were acquired from Gene Pharma (Shanghai, China). Phosphate buffered saline (PBS) and the assay kits to measure aspartate aminotransferase (AST), alanine aminotransferase (ALT), bicinchoninic acid (BCA), and hydroxyproline (HYP) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). CCl4 was from Sinopharm chemical reagent (Shanghai, China). Olive oil was bought from Agric-bemvig (Barcelona, Spain). FBS, trypsin, penicillin/streptomycin (Pen-Strep), and RPMI-1640 medium were purchased from Hyclone (Waltham, MA, USA). DMEM and LysoTracker™ Red were obtained from Gibco (CA, USA) and Beyotime (Shanghai, China). G418 and SDF-1 were bought from Life Technologies (Carlsbad, CA, USA). α-SMA, anti-TGFβ, anti-PCNA, anti-Col-III, and β-actin antibodies were purchased from Abcam (Cambridge, MA, USA). 3,3-Diaminobenzidine (DAB) kit was obtained from Boster biological (Hubei, China).
3
Comprehensive Protein Extraction and Analysis
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Total protein from fresh tissues or cells was lysed using a buffer containing RIPA buffer, 1% protein phosphatase inhibitor (Solarbio, Beijing, China), and 1% phenylmethanesulfonyl fluoride (PMSF, Solarbio, Beijing, China). Protein concentrations were quantified using a BCA Protein assay kit (Solarbio, Beijing, China). Proteins (20 micrograms) were separated on a 4–12% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Merck Millipore) at a current of 300 mA for 95 min. After transfer, the membranes were blocked and incubated with primary antibodies overnight at 4 °C. The primary antibodies used included anti-GAPDH (1:5000, Solarbio), anti-COL-I (1:2000, Abcam), anti-COL-III (1:2000, Abcam), anti- Transforming growth factor -β(TGF-β)(1:3000,Abcam),anti-TN-C (1:2000, Huaxingbio, Beijing), and anti-IL-6 (1:2000, Abcam). This was followed by a 1-hour room temperature incubation with IR Dye-conjugated secondary antibodies (1:5000, ZSGB-BIO, Beijing). The membranes were then imaged and quantified using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). To ensure robustness and reliability of data, the entire experimental procedure was repeated three times.
4
Quantitative Western Blot Analysis
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Proteins extracted from cells or tissues were quantified using Bradford Assay (Bio-Rad). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Bio-Rad). After blocking, the membranes were probed with primary antibodies and then incubated with specific horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Immunodetection was performed using enhanced chemiluminescence consistent with the manufacturer’s protocol (Thermo Fisher Scientific). Primary antibodies including anti-TAK1, anti-p-TAK1 (T187), anti-JNK, anti-p-JNK, anti-p38, anti-p-p38, anti-Smad3, anti-p-Smad3, anti-p65 and anti-β-actin were purchased from Cell Signaling Technology. Anti-p-TAK1 antibody (T184) was purchased from Thermo Fisher Scientific. Anti-Col I, Anti-Col III and anti-SIRT1 were purchased from Abcam. Anti-p20 antibody was purchased from BioVision (Milpitas, CA, USA). The band intensities were quantified and normalized to the corresponding controls. β-Actin was used as a loading control for internal correction.
5
Western Blot Analysis of Senescence and ECM Proteins
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The protein concentration in the supernatant was determined by BCA assay. Aliquots of 80 μg protein were separated in SDS-PAGE and transferred onto nitrocellulose membranes by an electric transfer (BIORAD Inc., USA). The membranes were blocked with 5% milk PBS-Tween20 (PBST) and incubated with primary antibody including: anti-Sesn1 (Abcam, USA), anti-Sesn2 (Santa Cruz Biotechnology, USA), anti-Sesn3 (Abcam, USA), anti-COL I (Abcam, USA), anti-COL III (Abcam, USA), anti-FN1 (Abcam, USA), and anti-Actin (Cell Signaling, USA) antibodies. They were then incubated with Alexa Fluor 700 antibodies (Molecular Probes, USA). Images were captured on an Odyssey Infrared Imaging System (LI-COR Biosciences, USA), and quantified using Odyssey v1.2.
6
Exosome Isolation and Characterization
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HCPT was purchased from Santa Cruz (CA, USA) and dissolved in dilute alkali solution followed by further dilution with normal saline or culture medium. TGF-β1 was purchased from Minneapolis (MN, USA). Fetal bovine serum (FBS) was obtained from Gibco (Carlsbad, CA, USA). High glucose Dulbecco’s modified Eagle’s medium (DMEM), alpha minimum essential medium (α-MEM), and penicillin/streptomycin were purchased from HyClone (Logan, UT, USA). Anti-CD9, anti-63, anti-ALIX, anti-TSG101, anti-calnexin, anti-CHOP, anti-Bcl-2, anti-α-smooth muscle actin (α-SMA), and anti-COL III antibodies were provided by Abcam Biotechnology (Cambridge, MA, USA); anti-Bax, anti-glucose regulated protein 78 (GRP78), and anti-β-actin antibodies were purchased from Cell Signaling Technology (CA, USA).
7
Renal Fibrosis Protein Profiling
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The proteins were isolated from the frozen kidneys and the concentration was calculated by the Bradford method. Immunoblotting examination was performed as previously described [21 ]. In this study, the primary antibodies used were anti-Ecadherin (1:1000,Abcam,UK), anti-ColIII (11,000, Abcam, UK),anti-caspase1. (1:1000, Abcam,UK), anti-TGFβR1 (1:1000,Abcam,UK), anti-Sirt1 (11,000, Abcam, UK), anti-NLRP3 (1:1000, Abclonal, China), anti-IL-1β (11,000,Abclonal,China), anti-Acetylated-lysine (1:500,Santa Cruz,USA), anti-ASC (1:500, Santa Cruz, USA), anti-TGF-β1 (1500,Santa Cruz,USA), anti-Smad3 (1,1000,CST,USA), and anti-Gapdh (12,000, Proteintech, USA). Protein bands were visualized using the chemiluminescence system (Tanon) for the required time. Quantitative analysis was performed using ImageJ software.
8
Immunohistochemical Analysis of Kidney Fibrosis
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Immunohistochemistry experiments were conducted on paraffin-embedded kidney tissue sections using anti-CoL I, anti-CoL III, and anti-α-SMA antibodies. The kidney tissue sections were dewaxed and dehydrated and then put in citrate buffer at 95 °C for 10–15 min, and then the tissue sections were incubated with anti-CoL I (Abcam), anti-CoL III (Abcam) and anti-α-SMA (Abcam) antibodies at 4 °C for about 12 h. The sections were incubated with the secondary antibody (Abcam). The DAB kit (Sangon Biotech) was applied to visualize the slices and counterstained the slices with hematoxylin (Sangon Biotech).
9
Western Blot Analysis of Fibrosis Markers
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To obtain total protein, tissues or HSF were lysed in RIPA buffer (Heart Biological Technology Co. Ltd, China) with Phenylmethanesulfonyl Fluoride (PMSF) added on ice. For western blot analysis, 30 μg of total protein was loaded onto a 10% Dodecyl Sulfate, Sodium Salt (SDS)-Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel, separated and transferred to a Polyvinylidene Difluoride (PVDF) membrane at 100 V for 60–80 min. The membranes were blocked with 5% nonfat milk and then incubated with primary antibodies at 4°C overnight. The next day, the membrane was washed and incubated with secondary antibodies (1:3000; Cell Signaling Technology (CST)) at 37°C for 2 h. Immunoreactive proteins were then visualized using a chemiluminescence system with an Enhanced Chemiluminescence (ECL) reagents kit (Millipore, USA). Protein band intensity was detected using the Fluor Chem FC system (Alpha Innotech, USA). Antibodies were as follows: anti-Col1 (rabbit, 1:1000; Abcam), anti-Col3 (rabbit, 1:3000; Abcam), anti-α-SMA (rabbit, 1:1000; Abcam), anti-EZH2 (rabbit, 1:1000; Abcam) and anti-GAPDH (Rabbit, 1:3000; Abcam).
10
Protein Extraction and Western Blot Analysis
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Fibroblasts were treated with lysis buffer. Total proteins of the cells were extracted using a Total Protein Extraction Kit (Keygen, Nanjing, China) supplemented with phenylmethylsulfonyl fluoride (PMSF), phosphatase inhibitors, and proteinase inhibitor. We measured protein concentration using an Enhanced Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime, Shanghai, China) and balanced them. After electrophoretic separation, transfer onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), and blocking procedures, proteins were incubated overnight with anti-α-SMA (Abcam, Cambridge, MA, USA, 1: 1000), anti-Col-1 (Abcam, Cambridge, MA, USA, 1: 1000), anti-Col-3 (Abcam, Cambridge, MA, USA, 1: 1000), anti-fibronectin (Abcam, Cambridge, MA, USA, 1: 1000), and anti-GAPDH (Proteintech, Rosemont, IL, USA, 1: 10 000). After washing with Tris-buffered saline-tween (TBST), the proteins were incubated with secondary antibody (YiFeiXue, Nanjing, China, 1: 10 000) for 1 h at room temperature. Proteins were then visualized and detected using an electrochemiluminescence (ECL) system.
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