Western Blot Analysis of NSCs and MSCs

The western blot assay was performed as described previously (Yu et al., 2016 (link)). NSCs and MSCs were prepared using RIPA buffer. The lysate protein concentrations were determined using a BCA protein assay kit (Beyotime Biotechnology, Nanjing, China). The protein samples were denatured in Laemmli buffer at 100°C for 10 min before electrophoresis, and were then subjected to 10% SDS-PAGE before being electrotransferred to polyvinylidene difluoride membranes with a standard transfer solution. After blocking with 10% nonfat milk, membranes were incubated with primary antibodies against β-tubulin III (1:1,000; Abcam, Cambridge, MA), MAP2 (1:1,000; Abcam, Cambridge, MA), Runx2 (1:1,000; Abcam, Cambridge, MA), BMP-2 (1:2000; ProteinTech Group), and OPN (1:2000; ProteinTech Group), and GAPDH antibody as a control (1:5,000; ProteinTech Group). Proteins were visualized by chemiluminescence using an ECL kit (Share-Bio, Jinan, China).