Tank blotting apparatus

The cells were treated with a Laemmli sample buffer (Cat. No. 161-0737, Bio-Rad, CA, USA) and heated to 100 °C for 5 min. The proteins were electrophoresed at 20 μg/lane on a denaturing sodium dodecyl sulfate-polyacrylamide gel (SDS–PAGE) and transferred to a nitrocellulose membrane (Whatman, GmbH, Hahne str., Germany) using a Bio-Rad tank blotting apparatus (Bio-Rad, Hercules, CA, USA). The membranes were probed with 1:1000–1:2000 dilutions of primary antibodies against p47 Phox (Santa Cruz Biotechnology), ERK1/2 (Cell Signaling Technology, Danvers, MA, USA), PKC_δ (Santa Cruz Biotechnology), AGE (Trans Genic Inc.), RAGE (Cell signalling), and β-actin (Sigma). The membrane was washed and incubated with a horseradish peroxidase-coupled goat anti-rabbit IgG (Santa Cruz Biotechnology). After washing the membranes thrice, the signals were detected with a WEST-one enhanced chemiluminescence (ECL) solution (Intron, Korea) using a Fujifilm LAS-3000 (LAS-3000, Fuji Photo, Tokyo, Japan). The band intensities were determined using Multi Gauge Version 3.0 software.

HNEpCs were seeded at 2.5 × 10⁵ cells/well in 6-well plates 24 h before treatment with 15 ng/mL IL-4/IL-13 (7.5 ng/mL of each) in the presence or absence of GJW, GJE, or Mito-TEMPO (Sigma-Aldrich) for 1 h. The HNEpCs were then lysed with Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and heated at 100 °C for 5 min. Next, 25–30 µg of these extracted proteins was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions. The separated proteins were then transferred to nitrocellulose membranes (Bio-Rad) using a tank blotting apparatus (Bio-Rad). The protein-blotted membranes were probed with specific primary antibodies against superoxide dismutase (SOD), catalase, uncoupling protein-2 (UCP2), phospho-p38, p-38, phospho-activating transcription factor-2 (ATF2), and β-actin (Cell Signaling Technology, Danvers, MA, USA; 1:1000 dilution), washed with tris-buffered saline containing 0.1% Tween-20, and incubated with horseradish peroxidase-linked secondary antibodies. After the membranes were washed three times, immunoreactivity was detected using the EzWestLumiOne enhanced chemiluminescence solution (Atto Corporation, Tokyo, Japan); protein bands were then visualized with ChemiDoc (Bio-Rad). Quantification of western blot images were performed using ImageJ 1.52a software (National Institutes of Health, Bethesda, MD, USA).