Protein Expression Analysis of DLL4/Notch Pathway

After treatment for 72 h, the cells were lysed and total proteins were extracted using RIPA buffer with protease inhibitors (Beyotime Biotechnology). Then, the protein concentration was determined by the BCA assay kit (Beyotime Biotechnology). Equal amounts of protein samples (40 μg) were separated by SDS-polyacrylamide gel electrophoresis and then transferred to PVDF membranes. The membranes were blocked in 5% fat-free milk, then cut according to the size of the target protein and incubated with primary antibodies against DLL4 (1:1000, Abcam), Notch (1:1000, Abcam), Hes1 (1:1000; Abcam), Bax (1:1000, Abways Technology), Bcl-2 (1:1000, Abways Technology), cleaved caspase-3 (1:1000, Cell Signaling Technology) and GAPDH (1:1000) (Beyotime Biotechnology) at 4 °C overnight and then incubated with secondary antibodies (1:4000, Beyotime Biotechnology) at room temperature for 1 h^{39 (link)}. Finally, the bands were exposed by ECL (Tanon, Shanghai, China).