*4.6. Immunoblot Analysis*

A549 and H1299 cells (1 <sup>×</sup> <sup>10</sup><sup>6</sup> ) were seeded onto a 60 mm dish. Cell lysates were prepared by extracting proteins with radioimmunoprecipitation assay (RIPA) lysis buffer (Millipore, Billerica, MA, USA) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific). Equal amounts of proteins were separated using SDS-PAGE on 8%–13% gels and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% skim milk in Tris-buffered saline-Tween 20 (TBST; 150 mM NaCl, 10 mM Tris, 0.2% Tween 20; Sigma-Aldrich), followed by overnight incubation with primary antibodies at 4 ◦C. Blots were developed using peroxidase-conjugated secondary antibody, and immunoreactive proteins were visualized using enhanced chemiluminescence reagents, according to the manufacturer's recommendations (Amersham, GE Healthcare, Buckinghamshire, UK). Protein bands were visualized using an ImageQuant LAS 4000 mini digital imaging system (GE Healthcare). Protein levels were analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA). Experiments were repeated at least three times.
