Western Blot Protein Analysis Protocol

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.