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3 protocols using anti collagen 3

1

Western Blot Analysis of Collagen and TGF-β1

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Protein samples were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). After blocking at room temperature in 5% w/v nonfat milk with TBST buffer (Tris-HCl 10 mM, NaCl 120 mM, and Tween-20 0.1%; pH 7.4) for 2 h, membranes were incubated overnight with the appropriate primary anti-collagen I, anti-collagen III, anti-TGF β1 (1 : 1000, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Smad2, anti-p-Smad2 (1 : 500, Bioworld Technology, St. Louis Park, MN), or anti-GAPDH (1 : 6000, Sigma-Aldrich, St. Louis, MO) antibodies, at 4°C, followed by horseradish peroxidase- (HRP-) conjugated secondary antibody at room temperature for 2 h. Proteins were visualized by enhanced chemiluminescence substrate (ECL, Pierce, Rockford, IL).
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2

Quantifying Fibrosis Severity: Western Blot Analysis

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In order to quantify the fibrosis severity, Western blot analysis of α-SMA, type I collagen and type III collagen expression was used. For each study group, 20 μg of protein from the cell lysates were separated by electrophoresis using the Biorad Western Blot MiniProtean system on acrylamide 12.5% gels. Then, the gels were transferred onto polyvinylidene difluoride (PVDF) membranes. Blots were washed, blocked and incubated with specific primary antibodies anti-α-SMA, anti-collagen I and anti-collagen III, at a 1:500 dilution, for 45 min at 4 °C (Santa Cruz Biotechnology, USA). Then, the membrane was incubated overnight with specific secondary antibodies, in 1:1000 dilution (Santa Cruz Biotechnology). Chemiluminescence was used for the immunolocalisation, in accordance with the protocol (Sigma-Aldrich, St. Louis, MO, USA). GADPH was used as a loading control for protein normalization.
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3

Western Blot Analysis of Fibroblast Proteins

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The fibroblasts were lysed in lysis buffer (Beyotime) and centrifuged for supernatant recovery. The protein concentrations were determined using a BCA kit (Beyotime). After being separated by 10% SDS-PAGE, the proteins (20 µg) were transferred onto nitrocellulose membranes (Millipore) and blocked with 5% non-fat dry milk for 1 h at room temperature. The membranes were then first incubated at 4°C overnight with anti-PTEN (1:200; cat. no. 9188), anti-DNMT1 (1:1,000; cat. no. 5032), anti-DNMT3a (1:1,000; cat. no. 49768), anti-DNMT3b (1:1,000; cat. no. 48488), anti-PI3K (1:1,000; cat. no. 4255), anti-p-PI3K (1:1,000; cat. no. 4228), anti-Akt (1:1,000; cat. no. 4685), anti-p-Akt (1:1,000; cat. no. 4056), anti-MMP-2 (1:1,000; cat. no. 87809) (all from Cell Signaling Technology), anti-α-SMA (1:350; cat. no. A03744, BosterBio), anti-MMP-9 (1:500; sc-21733), anti-caspase-3 (1:200; sc-271759), anti-collagen I (1:1,000; sc-376350), anti-collagen III (1:1,000; sc-271249) and anti-β-actin (1:1,000; sc-58673) (all from Santa Cruz Technology) antibodies and then with secondary antibodies [goat anti-rabbit IgG H&L (HRP) (1:5,000; ab6721, Abcam)] for 2 h at room temperature. The proteins were visualized using an ECL kit (Amersham Biosciences, Germany), and analyzed with the Bio-Rad ChemiDoc™ XRS+ System and Image Lab™ Software version 4.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
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