Determination of protein expression by western blot analysis

Protein extract (1 ml) was added into a culture dish, and a pipette was used to repeatedly blow the solution until the cells were fully pyrolyzed. The protein extraction buffer included 20 mM Tris-HCl (pH 7.5) and protease inhibitor (both from Beijing Solarbio Science & Technology Co., Ltd.). The solution was sucked out, placed in an Eppendorf tube, and centrifuged at 16,000 × g under 4°C for 15 min to collect the supernatant. The protein to be detected via the BCA method was separated using 15% polyacrylamide gel electrophoresis. Protein was transferred to a nitrocellulose (NC) membrane and was let to stand at room temperature for 1 h (blocked with 5% skim milk - phosphate-buffered saline (PBS) solution). The mass of protein loaded per lane was 15 µg. IL-8, SP-A, SP-B, caspase-9, caspase-3, Bax, Bcl2, TNF-α, IL-17 and IL-1β primary antibodies were added, and let to stand overnight at 4°C. The NC membrane was washed with PBS solution 3 times, and then HRP conjugated goat anti-rabbit secondary antibody was added, and let to stand for 1 h at room temperature. Finally, the NC membrane was washed with PBS solution, and visualized by the enhanced chemiluminescence method. ECL Western Blotting Substrate (Thermo-Fisher Scientific, Inc.) was used for visualization. The internal reference protein was β-actin and the relative expression level of the protein to be detected was calculated as (the gray value of the band to be detected)/(the gray value of β-actin protein band).