Western Blot Analysis of Tendon-Specific Genes

The protein expression of Mkx and other tendon-specific genes were measured with Western blot using the same condition as described previously [26 (link)]. Proteins were extracted using RIPA Lysis Buffer (Shanghai Weiao Biotechnology Co., Ltd., China). BCA protein reagent kit (Beijing Solarbio Science & Technology Co., Ltd., China) was used to measure the concentration. Thirty micrograms proteins were run on SDS-PAGE gels (8%) and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membranes were blocked with 10% skim milk at room temperature for 1 h and were incubated with anti-Mkx (1:200, Aviva Systems Biology, San Diego, USA), anti- Col-1a1 (1:400, Bioss, Beijing, China), anti-Collagen type III (Col-3a1, 1:400, Bioss, Beijing, China), anti-Dcn (1:400, Bioss, Beijing, China), and anti-Tnmd (1:400, Bioss, Beijing, China) primary antibodies solution and anti-β-Actin (1:1000, BOSTER Biological Technology Co., Ltd, China) primary antibody solution overnight at 4 °C. After reacting with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody or goat anti-mouse secondary antibody (BOSTER Biological Technology Co., Ltd, China) for 1 h at room temperature, proteins were detected with ECL hypersensitive chemiluminescence kit (Shanghai Weiao Biotechnology Co., Ltd., China) according to the manufacturer’s recommendations.