Enhanced chemiluminescence kit

The A549-F10, A549-mock and untransfected A549 cells were lysed using P0013 lysing agent (Beyotime Institute of Biotechnology, Suzhou, China), then cellular protein was separated by gel electrophoresis and transferred to membranes. The membranes were incubated with the anti-BAX, anti-caspase-3 and anti-β-actin primary antibodies, followed by incubation with the horseradish peroxidase-labeled rabbit anti-mouse secondary antibodies. The blots were visualized using an enhanced chemiluminescence kit (Kodak, Rochester, NY, USA) and the signal intensities were quantified using SensiAnsys software (Shanghai Peiqing Science and Technology, Co., Ltd., Shanghai, China).

Tissue samples from STANR, AF, and NP were rapidly removed and stored at −80°C until use. All tissue samples were homogenized in lysis buffer containing phenylmethane sulfonyl fluoride and 0.02% protease inhibitor cocktail. The homogenates were centrifuged at 14,000×g for 15 min at 4°C. Equivalent amounts of protein (40 μg) were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. Membranes were blocked with 5% milk for 1 h at room temperature and incubated with respective primary antibodies anti-CX3CL1 (1:1,000; Santa Cruz Biotechnologies, Inc) and anti-CCL2 (1:4,000; Abcam, Cambridge, UK) overnight at 4°C. The membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (1:5,000; Santa Cruz Biotechnologies, Inc). Bands were finally revealed using an enhanced chemiluminescence kit (Kodak, Rochester, NY, USA). The monoclonal antibody against β-actin (1:10,000; Sigma-Aldrich Co., St Louis, MO, USA) was used as a loading control. Bands area/intensity for all proteins were measured using Image J software (Wayne Rasband; National Institutes of Health, Bethesda, MD, USA). All the samples were routinely analyzed using Western blot repeated for at least three times, and the obtained results were averaged and calculated.