Anti tgf β1

Total protein was extracted from cells as previously described. Cells were lysed in RIPA buffer (Beyotime Biotechnology; China) containing 1% protease inhibitors (Roche Diagnostics, Mannheim, Germany) or 20 mM N-ethylmaleimide as appropriate (Sigma-Aldrich; St. Louis, MO, U.S.). The lysates were separated on an 8% or 12% SDS-PAGE gel and then transferred to nitrocellulose filter membranes. After being blocked in 5% non-fat milk, the membranes were incubated with the following primary antibodies: anti-PML (1:500 dilution; MBL, Nagoya, Japan), SUMO-1 (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.), SUMO-2/3 (1:500 dilution; Abcam, Cambridge, U.K.), UBC9 (1:500 dilution; Cell Signaling Technology, Beverly, MA, U.S.); RNF4 (1:500 dilution; Abcam, Cambridge, U.K.); anti-TGF-β1 (1:500 dilution; Cell Signaling Technology, Beverly, MA, U.S.) and anti-HERG (1:500 dilution; Alomone labs, Jerusalem, Israel). GAPDH (1:10,000 dilution; Research Diagnostics, Concord, MA, U.S.). Goat anti-rabbit (1:10,000 dilution; Alexa Fluor 700 conjugated; Molecular Probes/Life Technologies) served as the secondary antibody. Immunoblots were imaged using an LI-CORE Imaging System (LI-COR Biosciences, Lincoln, NE, U.S.) and Odyssey software was used for quantification of bands normalized to GAPDH.

The spinal cords were freshly collected and homogenized in tissue lysis buffer. The lysates were centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatant was collected and stored at − 80 °C. The Bradford method (Bradford, 1976) was performed to quantitate protein concentration. SDS-PAGE electrophoresis was performed by using a 12% polyacrylamide gel and 30 μg/well protein, and separated protein was then transferred for 45 min onto a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in TBST (20 mM Tris base, 130 mM NaCl, 0.1% Tween-20) and then incubated overnight at 4 °C with primary antibodies: anti-IL-20, anti-TGF-β1 (Cell Signaling), and anti-β-actin (Proteintech®). After primary antibody incubation, the membrane was washed with TBST. Following the wash steps, the membrane was incubated with species-specific horseradish peroxidase-labeled (HRP) secondary antibody for 1 h at room temperature. Following the final wash steps, the membrane was developed with enhanced chemiluminescent substrate and visualized on film (FUJI MEDICAL X-RAY FILM, FUJIFILM, Japan). The protein signals were digitalized by a scanner and analyzed by the Image-pro plus 4.5.1 software.

Total protein was extracted using RIPA lysis buffer (Beyotime) and quantified using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Subsequently, 30 µg protein sample was separated by 10% SDS‐PAGE and transferred to PVDF membranes. Membranes were blocked in 5% nonfat milk for 2 h at room temperature and incubated with primary antibodies overnight at 4°C. The primary antibodies for western blot analysis were as follows: anti‐GAPDH, anti‐TGF‐β1, anti‐SMAD1, anti‐pSMAD1, anti‐SMAD5, and anti‐pSMAD5 (all from Cell Signaling Technology). Membranes were then incubated with horseradish peroxidase‐labeled secondary antibody (Cell Signaling Technology) for 1 h at room temperature. Protein signals were visualized using chemiluminescence reagent (Beyotime) and captured using Invitrogen iBright 1500 (Thermo Fisher Scientific). Results were analyzed using ImageLab software (Version 5.0; Bio‐Rad Laboratories).

Mice were sacrificed for histopathologic evaluation 14 weeks after irradiation. The skin and soft tissue of the irradiated leg was formalin-fixed, paraffin-embedded, cut into 4 μm sections, and stained with hematoxylin-eosin using standard procedures. To detect collagen accumulation in fibrotic tissues, Masson's trichrome staining was performed according to the manufacturer's protocol (Diagnostic BioSystems, Pleasanton, CA). For the immunohistochemical analysis, deparaffinized slides were incubated with anti-TGF-β1 (Cell Signaling), anti-PAI-1 (Santa Cruz Biotechnology), anti-SMA (abcam), anti-MMP2 (Santa Cruz Biotechnology), anti-MMP9 (Santa Cruz Biotechnology), anti-p300 (Santa Cruz Biotechnology), anti-CBP (Santa Cruz Biotechnology), and anti-acetylated p65 (Cell Signaling) primary antibodies, and subsequently with horseradish peroxidase-conjugated secondary antibodies. The DAB⁺ chromogen were used for signal detection (Dako Real Envision detection kit; Dako, Glostrup, Denmark). Representative images of the brown staining within fibrotic regions were captured and evaluated.