Anti tnc

To examine the production of proteins in the BMP12/Smad1/5/8 pathway during tenogenic differentiation, TSCs or EGR1-TSCs were cultured for 0 h, 12 h, 1 d, 3 d, 7 d, and 14 d. Cells were washed twice with PBS and lysed in lysis buffer containing a mixture of proteinase inhibitors (Thermo Fisher Scientific Inc., Rockford, IL, USA). To determine levels of tenocyte-related proteins and those in the BMP12/Smad1/5/8 pathway during tendon healing, tendon tissues were digested in a buffer containing 8 M urea, 50 mM Tris-HCl (pH 8), 1mM dithiothreitol, and 1 mM EDTA. Total protein concentrations were measured using a BCA protein assay kit (Thermo Fisher Scientific Inc.), and equal amounts of extracted proteins (30 µg/lane) were resolved by SDS-polyacrylamide gel electrophoresis. Proteins were then transferred on to polyvinylidene difluoride membranes, and membranes blocked by incubating with 5% non-fat milk containing 0.1% TBS-Tween for 1 h at 20°C. The membranes were then incubated sequentially with primary and secondary antibodies. The following primary antibodies were used: anti-BMP12, anti-phospho-Smad1/5/8, anti-Smad, anti-SCX, anti-TNMD, anti-TNC, or anti-Collagen I (all from Santa Cruz Biotechnology). β-actin was used as an internal control. Proteins were visualized and images captured using a LiCoR Odyssey imager (LI-COR Biosciences, Lincoln, NE, USA).