Ab264043

The total protein of cells was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime, Nanjing, Jiangsu, China). The protein concentration was assessed using the bicinchoninic acid (BCA) protein assay kit (Beyotime). The samples were loaded for electrophoresis. Then, the protein was transferred to the polyvinylidene fluoride (PVDF) membranes using the wet method, electric transferred in a cold chamber at a voltage of 70 V at 4 °C for 2 h, and the PVDF membranes were removed and blocked with 5% skim milk-Tris buffered saline-Tween20 (TBST), and incubated at room temperature for 1 h. After blocking, the membranes were placed into the incubation box and cultured with rabbit anti-mouse primary antibodies anti-APT1 (1:1000, 25 kDa, ab91606, Abcam), anti-OCN (1:500, 11 kDa, ab93876, Abcam), anti-RUNX2 (1:1000, 57 kDa, ab236639, Abcam), anti-Osterix (1:1000, 47 kDa, ab209484, Abcam), anti-BMPR1a (1:1000, 60 kDa, ab264043, Abcam), BMP (1:1000, 44 kDa, ab214821, Abcam), p-Smad (1:1000, 52 kDa, ab92698, Abcam) at 4 °C overnight. Subsequently, the samples were eluted with TBST and incubated with secondary antibody goat anti-rabbit IgG (1:20000, ab6721, Abcam) at room temperature for 1 h. With GADPH (1:5000, 37 kDa, ab9485, Abcam) as the internal parameter, chemiluminescence method was employed for detection and gray analysis.