Cells were lysed with radioimmunoprecipitation (RIPA) buffer (Biosesang, Seoul, Korea) containing 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 μg/mL pepstatin, and 10 μg/mL leupeptin for 20 minutes at 4°C, scraped with a cell scraper, and centrifuged at 15,000g, for 15 minutes, at 4°C. Cell lysates were boiled in Laemmli's sample buffer (Bio-Rad, Hercules, CA, USA) for 5 minutes. Proteins were separated by SDS-PAGE (Bio-Rad) on 6.5% gels for mucin and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked overnight at 4°C in 3% BSA or 5% nonfat dry milk in buffer containing 10 mM Tris-HCl (pH 8.0; Sigma-Aldrich), 150 mM NaCl (Sigma-Aldrich), and 0.05% Tween-20 (Sigma-Aldrich), and then incubated overnight with primary antibody for MUC1 (sc-7313, 1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), MUC4 (ab60720, 1:1000; Abcam, Cambridge, MA, USA), MUC5AC (ab24070, 1:200; Abcam), MUC16 (ab10033, 1:300; Abcam), P2Y1 (1:1000; Abcam), P2Y2 (1:1000; Abcam), P2Y4 (1:1000; Abcam), P2Y6 (1:1000; Abcam), or β-Actin (1:1000; Santa Cruz Biotechnology), followed by incubation with peroxidase-labeled anti-mouse IgG secondary antibody (1:3000; KPL, Gaithersburg, MD, USA) or peroxidase-labeled anti-rabbit IgG secondary antibody (1:3000; KPL).
β-Actin served as a loading control. Cells exposed to 10 μg/mL lipopolysaccharide (LPS; Sigma-Aldrich) for 1 hour served as a positive control. Immunoreactive bands were visualized using an enhanced chemiluminescence Western blotting kit (Amersham Pharmacia, Piscataway, NJ, USA). Results are representative of at least three independent experiments.