Western Blot Analysis of Osteogenic Signaling

Western blot analysis was performed as previously described [65 (link)]. Briefly, proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA), and the membranes were blocked for 1 h at room temperature and incubated overnight at 4 °C with the primary antibodies as follows: AKT (1:1000, #4691, Cell Signaling Technology, Beverly, MA, USA), p-AKT (1:1000, #4060, Cell Signaling Technology), β−actin (1:1000, #sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BMP2 (CUSABIO, #CSB-PAO9419AORb, Houston, TX, USA), Smad1/5/8 (1 : 1000, #sc-6031-R, Santa Cruz Biotechnology), p-Smad1/5/8 (1:2000, 13820, Cell Signaling), GSK3β (1:1000, #12456, Cell Signaling), p-GSK3β (1:1000, #9336, Cell Signaling), RUNX2 (1:1000, #12556, Cell Signaling Technology), and Wnt3a (1:1000, #2721, Cell Signaling). Then, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1:10,000, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. The protein signals were monitored in the ProteinSimple detection system (ProteinSimple Inc., Santa Clara, CA, USA).