Sdf 1

The cells and tissue specimens were lysed with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing phenylmethanesulfonyl fluoride (Beyotime) and a protease inhibitor (CoWin Biosciences). The protein concentration was determined using a bicinchoninic acid protein assay kit (Beijing Solarbio Science and Technology Co., Ltd. Beijing, China). Each sample with equal amounts of protein were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. After running 1–2 h under 120V, the protein was transferred onto a polyvinylidene difluoride membrane. Next, the membranes were blocked with 5% not-fat milk for 1 h at room temperature and incubated with primary antibodies c-Myc, HIF-1α, CXCR4, SDF-1, vimentin, E-cadherin, and β-actin overnight at 4°C (Affinity Biosciences). Subsequently, the membranes were washed 3 times with tris-buffered saline and Tween-20 and incubated with a horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technology, Beverly, MA, USA). Finally, the membranes were treated with a chemiluminescence reagent and exposed to X-ray. The band intensities were quantified using image J software (National Institutes of Health, Bethesda, MD, USA). Relative protein expression was quantified using control protein β-actin.

The protocol and procedure for Western blot were performed as described in previous reports [40 (link)]. For each sample, the 30 μg extracted total protein was loaded onto a 10–15% SDS/PAGE gel. The gel-separated protein was then transferred to a PVDF membrane (Millipore) and incubated with the primary antibodies of anti-transforming growth factor-β (TGF-β, Abcam), anti-SMAD2/3 (Abcam), anti-collagen type II (COL II, Abcam), anti-SOX-9 (Abcam), anti-collagen type X (COL X, Abcam), anti-Indian hedgehog (IHH, Affinity, OH, USA), anti-matrix metalloproteinase 13 (MMP 13, Affinity), anti-stem cell-derived factor 1 (SDF-1, Affinity), anti-vascular endothelial growth factor (VEGF, Affinity), and anti-β-actin (Invitrogen) at 37 °C for 2 h, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Protein expression was visualized, and the values were normalized against β-actin.