Protease and phosphatase inhibitors

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 lysed in buffer containing protease and phosphatase inhibitors (Dingguo, Beijing, China). Proteins were quantified, separated by electrophoresis on 8% polyacrylamide gels, and then transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked and probed with antibodies for integrin α5 (Millipore), integrin β1 (Millipore), ERK1/2 (Cell Signaling, Danvers, MA, USA), phospho-ERK1/2 (Cell Signaling), FAK (04–591; Millipore), Akt (Cell Signaling), p-Akt (Cell Signaling), GSK-3β (Cell Signaling), p-GSK-3β (Cell Signaling), β-catenin (Cell Signaling), and GAPDH (Sigma-Aldrich) overnight at 4 °C, followed by secondary antibodies on the next day. Immunoreactive bands were visualized by chemiluminescence.