Protein Extraction and Western Blot Analysis

RIPA (Sigma-Aldrich, USA) reagents were used to extract proteins from hAECs. Protein concentration was determined using a bicinchoninic acid (BCA) kit (Sigma-AldrPPROM, USA). The extracted proteins (20 µg/lane) were then separated using a 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE gel), transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA), and blocked with 5% skimmed milk for 2 h. The membranes were then incubated with primary antibodies including ki67 (abcam, 1:1000), ELK1 (abcam, 1:800) and GAPDH (Bioss, 1:2000) at 4°C overnight, followed by incubation with the corresponding goat anti-mouse antibodies and goat anti-rabbit IgG antibodies for 1 h. Subsequently, the membranes were stained with an ECL western blotting kit (ab133406, Abcam, USA). GAPDH was used as a loading control. Finally, the protein bands were visualized using an enhanced chemiluminescence system (Thermo Fisher Scientific, USA) [20 (link)].