Western blot analysis

The expression quantity of various proteins was determined in human lung cancer A549 cells using western blotting according to standard procedures. In short, total protein from untreated or treated cells was extracted in RIPA lysis buffer. The same amount of protein (30 μg) from each group were separated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Bio‐Rad Laboratories, Hercules, USA). Each membrane was incubated with a specific primary antibody (1:1000) at 4°C overnight after blocking with 5% skim milk at room temperature for 1 h. After three washes with washing buffer (20 mmol/L Tris‐HCl, 500 mmol/L NaCl, and 0.1% Tween 20), each suitable secondary antibody was incubated in the membrane at room temperature for 2 h. An ECL Advanced Western Blot Detection Kit (Thermo Fisher, Waltham, USA) was used to visualize the specific protein bands.