Tgf β 3711 s

The oligo-fucoidan powder was obtained from Hi-Q Marine Biotech International (Taiwan) and prepared by enzyme hydrolysis of original fucoidan. In brief, 5 g of fucoidan from Sargassum hemiphyllum was suspended in 125 mL distilled water at 55 °C with at 700 rpm stirring speed followed by addition of glycolytic enzyme at a concentration of 1 mg/g fucoidan for 6 h. After centrifugation at 10,000 × g for 20 min at 4 °C, the supernatants were passed through a 30 kDa molecular weight cut-off membrane (ProStream™ PP, TangenX Technology Co., Boston, MA, USA) and the filtrate was further passed through a 1 kDa molecular weight cut-off membrane to obtain the oligo-fucoidan with the average molecular weight of 800 Da. Oligo-fucoidan was dissolved in double-distilled H₂O and filtered using 0.22-μm sterile filters (Merck Millipore, Darmstadt, Germany). Hyaluronan (molecular weight 0.6–1.1 MDa) and all other chemicals of reagent grade were obtained from Sigma (St. Louis, MO, USA). Recombinant mouse osteopontin and anti-osteopontin (AF808) were obtained from R&D Systems (Minneapolis, MN, USA). Antibodies against fibronectin (sc-9068) and Smad2/3 (sc-8332) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against αSMA (4968 s), β-catenin (8480 s), CD44 (3570 s), p-Smad2/3 (8828 s), and TGF-β (3711 s) were obtained from Cell Signaling Technology (Danvers, MA, USA).