Western Blot Analysis of Protein Expression

WB was performed in accordance with the method described by Luo et al. [10 (link)]. Total cell extracts were resolved by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis. The protein was then electrophoretically transferred to polyvinylidene fluoride membranes. The membranes were incubated overnight at 4 °C with diluted (1:1000) rabbit anti-human pendrin (bs-19817R; Bioss, Beijing, China), rabbit antihuman HNE (ab68672; Abcam, Cambridge, UK), rabbit anti-human EGFR (bs-0405R; Bioss), β-actin polyclonal antibodies (GB13001-1; Servicebio, Wuhan, China), and glyceraldehyde-3-phosphate dehydrogenase polyclonal antibodies (GB13002-m-1; Servicebio, Wuhan, China). Membranes were then washed and incubated in goat anti-rabbit antibodies (GB23303; Servicebio, Wuhan, China) for 1 h. Finally, membranes were washed three times with Tris-buffered saline–Tween. Under the condition of avoiding light, the A and B solution (Beyotime Biotech, Shanghai, China) in the luminescent liquid reagent shall be mixed evenly by 1:1. After incubation for an appropriate time, membranes were exposed in the chemiluminescence instrument (Bio-Rad company, Hercules, CA, USA).