Western blot analysis of phosphorylated (p)-ERK and ERK

Prior to western blot analysis, A549 cells were treated with CCL20 (500 ng/ml; R&D Systems, Inc.) for 0, 5, 15, 30 and 60 min. A549 cells with various treatments were harvested and lysed on ice in RIPA buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, pH 8.0, 0. 2 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride and 0.5 % NP-40; cat. no. 89900; Thermo Fisher Scientific, Inc.) containing a protease inhibitor. The total protein concentration was measured using the BCA method. Proteins (60 µg per lane) were subjected to SDS-PAGE using a 10% gel, and blotted onto nitrocellulose membranes (Hybond ECL; GE Healthcare, Chicago, IL, USA). Membranes were blocked by incubation in TBS containing 5% nonfat dry milk and 0.1% Tween-20 for 2 h at room temperature and then incubated overnight at 4°C with rabbit anti-human p-ERK antibodies (dilution, 1:1,000; cat. no. sc-101760; Santa Cruz Biotechnology, Inc.), ERK antibodies (dilution, 1:1,000; cat. no. sc-292838; Santa Cruz Biotechnology, Inc.) and β-actin (dilution, 1:1,000; cat. no. sc-130656; Santa Cruz Biotechnology, Inc.). Blots were then washed with TBS/Tween-20 three times and incubated at room temperature for 1 h with horseradish peroxidase-conjugated goat anti-rabbit antibodies (cat. no. ab97051; Abcam, Cambridge, UK). The bands were detected on an X-ray film following application of Pierce ECL Western Blotting Substrate Thermo Fisher Scientific, Inc.). Relative optical density of all bands was analyzed using GelPro Analyzer 4.0.