Dieckol, a Component of Ecklonia cava, Suppresses the Production of MDC/CCL22 via Down-Regulating STAT1 Pathway in Interferon-? Stimulated HaCaT Human Keratinocytes

Kang, Na-Jin; Koo, Dong-Hwan; Kang, Gyeoung-Jin; Han, Sang-Chul; Lee, Bang-Won; Koh, Young-Sang; Hyun, Jin-Won; Lee, Nam-Ho; Ko, Mi-Hee; Kang, Hee-Kyoung; Yoo, Eun-Sook

HaCaT cells were washed twice with ice-cold phosphate buffered saline (PBS), and then disrupted in lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonident P-40, 2 mM ethylenediamine tetraacetic acid (EDTA), 1 mM ethylene glycol bis (2-aminoethylether)-N,N,N_,N_-tetraacetic acid (EGTA), 1 mM NaVO3, 10 mM NaF, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, and 25 mg/mL leupeptin) on ice for 30 min. Cell lysates were centrifuged at 15,000 rpm for 15 min at 4 o C, and the supernatants were used for Western blotting. The total protein concentration of each sample was quantified via the Bio-Rad assay method (Bio-Rad). Extracts containing 30 mg of protein were loaded next to a prestained protein-mass ladder (Bio-Rad) on a NuPAGE 4-12% bis-Tris gel (Invitrogen, Carlsbad, CA, USA). The proteins were elec troblotted onto a polyvinylidene difluoride (PVDF) membra ne by using an iBlot gel transfer device (Invitrogen). The membrane was blocked with blocking buffer (5% skim milk in Tween 20-Tris buffered saline (TTBS)) for 1 h at room temperature, followed by overnight incubation at 4°C with the appropriate primary antibodies (anti-phospho-STAT1, anti-STAT1, anti-phospho-ERK, anti-ERK, anti-phospho-JNK, anti-JNK, anti-phospho-p38, anti-p38, and anti-β-actin antibodies). All antibodies were diluted in 1% bovine serum albumin (BSA) in TTBS buffer. After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-primary antibody host immunoglobulin G (IgG) diluted 1 : 5000 for 1 h at room temperature. After washing again, immunoreactive bands were visualized with a western blot detection system (iNtRON Biotechnology) according to the manufacturer's instructions.