Hedgehog Signaling and Cartilage Proteins

Expression of hedgehog signalling molecules and cartilage-related proteins was determined by western blotting as described previously [14 (link), 23 (link)]. Antibodies recognising Ptc (Aviva Systems Biology, San Diego, CA, USA; 1:500), Smo (Aviva Systems Biology; 1:1,600), Gli1 (Biorbyt, Cambridgeshire, UK; 1:1,000), Sox9 (OriGene, Rockville, MD, USA; 1:500), collagen II (Novus Biologicals, Littleton, CO, USA; 1:200), ACAN (Novus Biologicals; 1:100), collagen X (Abcam; 1:500), RUNX2 (Abcam; 1:500), ALP (Abcam; 1:500), PPAR-γ (Santa Cruz Biotechnology; 1:1000) and GAPDH (Novus Biologicals; 1:2000) were used as primary antibodies. The secondary antibody (1:2000) was purchased from Thermo (Waltham, MA, USA). GAPDH was used as an internal control protein.