Complete miniprotease inhibitor cocktails

The colons were removed from the saline-, S.C.-, S.C./pLJD-, or S.C./pLJD-CMV-mIL19-treated DSS colitis-treated mice at day 3 and 5. After diarrhea and fecal blood analyses, the specimens were subjected to PBS supplemented with complete miniprotease inhibitor cocktails (Roche Molecular Biochemicals, Mannheim, Germany). After homogenization, the samples were centrifuged at 16,000× g for 10 min at 4 °C to precipitate the insoluble cellular debris, and the supernatant was stored at −80 °C until analysis. The concentrations of TNF-α, IL-10, IL-6, and IL-1β were determined using the mouse Quantikine enzyme-linked immunosorbent assay (ELISA) kits (TNF-α and IL-10, Preprotech) (IL-6 and IL-1β, R&D Systems, Minneapolis, MN, USA).

Human primary keratinocytes were pretreated with SFII for 30 min, then stimulated with 10 μg/ml poly(I:C) for 3 h. Cells were washed with PBS, lysed with 1×TNE buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, and 1% NP-40) containing complete mini protease inhibitor cocktails (Roche Applied Science, Indianapolis, IN, USA) and phosphatase inhibitor cocktails (Sigma-Aldrich), and heated at 95°C for 5 min. Equal amounts of cell lysate were separated on a 10% SDS-polyacrylamide gel by electrophoresis and transferred onto a nitrocellulose membrane (GE Healthcare, Chicago, IL, USA). Equal protein transfer was confirmed by Ponceau S staining (ELPIS Biotech, Daejeon, South Korea). The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween^® 20 (TBS-T) and probed overnight with the indicated primary antibodies at 4°C. After washing with TBS-T, the membranes were probed with HRP-conjugated secondary antibodies for 1 h at 25°C and visualized using WestGlow™ PICO PLUS or FEMTO chemiluminescent substrates (Biomax Co., Ltd., Seoul, Korea) using a CCD imaging system (Amersham Imager 680, GE Healthcare). Only the data with unsaturated signals were used in the analysis.