Ab24071

Total protein from 16HBE cells was extracted using RIPA lysis buffer (Elabscience, Wuhan, China). After protein concentrations were determined by BCA Protein Assay Kit (Takara, Japan), equal amount of protein for each sample was resolved with SDS-PAGE (10%) and then immobilized onto PVDF membranes (Roche, Basel, Switzerland). The membranes were soaked in 5% non-fat dried milk in PBS and incubated with antibodies against MUC5AC (1:1,000, ab24071, Abcam), p65 (0.5 µg/mL, ab16502, Abcam), Lamin B1 (0.1 µg/mL, ab16048, Abcam), and β-actin (1:5,000; ab8226, Abcam) overnight at 4 °C respectively, and then incubation with corresponding secondary antibody at room temperature for 60 min. The blots were revealed with an ECL kit (Abcam) and analyzed by Image Software (NIH, Bethesda, MD, USA).

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.