Western blot analysis

Following treatment, A549 cells were harvested and lysed using radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.) supplemented with PMSF (1 mM). The whole cell lysates were centrifuged at 12,000 × g for 15 min at 4°C, and protein content in the supernatant was determined by the bicinchoninic acid protein assay kit (Applygen Technologies, Inc.). Proteins samples (30 µg per lane) were separated by 10% SDS-PAGE and transferred to PVDF membranes at 4°C for 90 min. After blocking with 5% non-fat milk at room temperature for 60 min, membranes were incubated with the following primary antibodies overnight at 4°C: Anti-PI3K p85α (cat. no., ab191606; 1:1,000); anti-phosphorylated (p)-PI3K p85α (Y607; cat. no., ab182651; 1:1,000); anti-AKT1/2/3 (cat. no., ab179463; 1:10,000); anti-p-AKT1 (Ser473; cat. no., ab81283; 1:1,000); and anti-GAPDH (cat. no., ab181602; 1:10,000). All primary antibodies were purchased from Abcam. The membranes were then probed with IgG-horseradish peroxidase-conjugated goat anti-rabbit (cat. no., ab2057; Abcam; 1:10,000) and goat anti-mouse (cat. no., ab6789; Abcam; 1:10,000) secondary antibodies for 1 h at room temperature. Finally, the proteins were detected using ECL Enhanced Chemiluminescent kit (Thermo Fisher Scientific, Inc.). Densitometric analysis was performed using ImageJ 1.44p software (National Institutes of Health).