Western Blot Analysis of Protein Expression

After OSM and STAT3 inhibitor treatment, the total proteins were extracted by RIPA buffer (Pierce, Rockford, IL) on ice. Equal quantities of protein samples were separated by electrophoresis on 12% SDS-PAGE polyacrylamide gels. Then, the samples were electro-transferred to PVDF membranes (0.22 μm, Millipore) using a wet transfer apparatus (Bio‐Rad) and blocked with 5% BSA in PBS for 1 h at room temperature. The membranes were incubated overnight at 4 °C with primary antibodies of CD44, FN1, CHI3L1, CD24, DLL3, OLIG2 and β-ACTIN (Abcam), respectively. After that, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (#HA1001, huabio, 1:5000) at room temperature for 1 h [30 (link)]. The immobilon reagents (Millipore) was used for the visualization and detection of antibody-antigen complexes. The band intensity was measured by ImageJ software.