Ta346894

Protein was extracted by RIPA cell lysate (containing PMSF) as previously described [11 (link)]. Protein samples were heated at 100°C for 10 minutes, and the protein concentration was quantified using bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was processed and 25 μg of protein was loaded onto nitrocellulose membrane. The membrane was blocked with 5% defat milk in PBST at room temperature for two hours. The primary antibodies against MUC5AC (1: 1,000, ab24071, Abcam, USA), JNK1 (1: 1,000, ab199380, Abcam, USA), and actin (1: 500, TA346894, Zsbio, Beijing, China) were incubated with the membrane overnight at 4°C. After rinsing (three times, 10 minutes each time), the secondary antibody (1: 10,000, ab131368, Abcam, USA) was incubated with the membrane for two hours at room temperature. Chemiluminescent substrate detection reagent was applied to assist the staining. Target band was analyzed by ImageJ software for grayscale analysis.