Quantification of PI3K/AKT/mTOR signaling pathway by western blotting

After centrifugation at 2000 r/min at 4 °C for 10 min, the A. castellanii were collected and lysed with RIPA lysate containing 1% protease inhibitor for 20 min on ice and then sonicated for 5 min. The protein in the supernatant was collected after centrifuging at 12,000 r/min at 4 °C for 15 min. The concentration of protein was determined using BCA kit according to the manufacturer’s instructions. After denaturation at 100 °C for 10 min, protein fractions (30 μg) were isolated by 12% SDS-PAGE gel electrophoresis and transferred onto PVDF membranes. The PVDF membranes were blocked with 5% (w/v) skim milk for 1 h at room temperature and then incubated with LC3B (1:1000), AKT(1:1000), mTOR (1:1000), p-AKT (1:1000), p-mTOR (1:1000) and β-actin (1:2000) antibodies overnight at 4 °C. After washing with TBST, the membranes were incubated with HRP goat anti-rabbit IgG (1:3000) and HRP goat anti-mouse IgG (1:3000) for 1 h at 37 °C. The protein bands were developed with ECL luminescent agent and analyzed using Image J software. The experiment was repeated three times.