Bmprii

The proteins were extracted from cells using buffer containing protease and phosphatase inhibitors (Dingguo, Beijing, China). The proteins were separated by electrophoresis on 8% polyacrylamide gels, and then transferred to polyvinylidene fluoride (PVDF) membranes. Afterwards, the membranes were blocked and probed with antibodies for BMPRIA (Abcam), BMPRIB (Abcam), BMPRII (Abcam), GAPDH (CST), Nestin (CST), βII-tubulin (CST), GSK-3β (CST), and FAK (CST) overnight at 4°C. The secondary antibodies were incubated with the membranes on the next day. Finally, the protein bands were visualized using chemiluminescence.

Cells were washed three times with ice-cold PBS and fixed with 4% paraformaldehyde-PBS. After 15 min of incubation with 0.1% Triton-PBS, the cells were blocked with 1% bovine serum albumin-PBS. Then, the following primary antibodies were used: (1) for hNSCs characterization, mouse anti-nestin polyclonal (1:1000, Santa Cruze, USA); (2) for hNSCs differentiation, the primary antibodies including Rabbit anti-glial fibrillary acidic protein (GFAP) (1:1000, Santa Cruze, USA), Rabbit anti-oligophrenin-4 (O4) (1:1000, Santa Cruze, USA), and mouse anti-Tuj-1(1:1000, Santa Cruze, USA) were used; (3) for hGSCs characterization, the hGSCs sphere were then incubated with mouse anti-human CD133 polyclonal antibody and mouse anti-nestin polyclonal (1:1000, Santa Cruze, USA); (4) for detection of the expressions of BMPs signaling molecules, mouse anti-human BMPR-IA, BMPR-IB and BMPR-II (Abcam, UK) were used, as well as mouse anti-Smad1 antibody, rabbit anti-phospho-Smad1 antibody (Cell signaling, USA); and (5) for examination of the proliferation and differentiation of hGSCs, mouse anti-Ki67 polyclonal antibody and mouse anti-GFAP monoclonal antibody (Abcam, UK) were used.

Total cellular protein was extracted in RIPA lysis buffer (Solarbio) and obtained by centrifugation at 4°C for 15 min at 12,000 rpm, and protein concentrations were quantified by a BCA kit (Solarbio). The samples were separated by 10% SDS‐PAGE (Solarbio) and transferred to a PVDF membrane. After being blocked with 5% skimmed milk in PBS for 2 h, the membrane was incubated separately overnight with the following rabbit antibodies at 4°C: acetylated α‐tubulin, γ‐tubulin, α‐tubulin, collagen type 1 (COL‐1), osterix (OSX), BMP‐2, RUNX‐2, BMPRII, BMPRIA, BMPRIB, p‐Smad1/5/8, Smad1/5/8, IFT88 or GAPDH (all from Abcam, 1:1000). After probing with a secondary antibody (goat anti‐rabbit HRP‐conjugated IgG at 1:1000, Abcam), immunoreaction signals were visualized with the ECL chemiluminescence reagent (Solarbio).