4.12. Western Blotting.
The following procedures were used to lyse the cells: Cold PBS was used to wash the cells for three times. Cells were then gathered using an enzyme-free dissociation buffer, and then lysed using an ice-cold RIPA buffer contained phosphatase and protease inhibitors. Lysates were performed on 10% SDS-Tris glycine gels for 2 h and then transferred to PVDF membranes (45 μm) for 1 h. The PVDF membranes were blocked using 5% w/v skim milk for 2h. The primary antibody was then incubated on the membranes overnight at 4 °C. Secondary antibody was incubated for 2h at 4 °C. Pierce ECL Western Blotting Substrate was used to view the signals, and ImageJ was used to quantify the signals. GAPDH was measured as a control.