Anti lcn2

Cell and tissue samples were lysed using RIPA buffer (Sigma-Aldrich, Saint Louis, MO) supplemented with protease inhibitor mixture (Roche Diagnostics). Protein concentrations were measured using the bicinchoninic acid (BCA) method. Equal amounts of proteins were loaded and separated on 10% SDS-PAGE, transferred to a nitrocellulose membrane and probed with indicated antibodies. Blots were visualized by enhanced chemiluminescence (Thermo Scientific, Rockford, IL). The following antibodies were used in this study: anti-Gbp1, anti- NFκB and anti-Tom20 (Santa Cruz); anti-phospho-NFκB, anti-phospho-p53, anti-p53, anti-phospho-AMPK, anti-AMPK, anti-Parkin, anti-p62, anti-LC3I/II, anti- β-actin and anti-tubulin (Cell Signaling Technology); anti-Lcn2 (R&D System, Minneapolis, MN).

Six-to-eight-week-old male C57BL/6N mice were obtained from Orient Bio Inc. (Seongnam, Korea), and housed in an air-conditioned room at 22–24 °C and 50–60% humidity with a 12 h light/dark cycle. JC1-40 was administered at a dose of 20 mg/kg/day in 0.5% carboxymethyl cellulose by oral gavage for 3 days, and mice were then injected with 100 mg/kg DEN (Sigma-Aldrich, St. Louis, MO, USA) by intraperitoneal injection. Two days after DEN treatment, the mice were sacrificed, liver tissues were excised rapidly, and portions of the liver were stored for further analysis of protein and mRNA or fixed in 10% formalin for histopathological analysis. Animal experiments were approved and conducted in accordance with guidelines of Seoul National University Animal Care and Use Committee (permission number SNU-130305-1).
For histological examination, 3 μm sections of paraffin-embedded tissues were stained with hematoxylin and eosin (H&E). Immunohistochemistry was performed using anti-IL-6Rα (Santa Cruz Biotechnology), anti-CXCL1 (Novus Biologicals, Littleton, CO, USA), anti-LCN2 (R&D Systems) anti-γH2AX (Abcam, Cambridge, MA, USA) antibodies. Serum concentrations of IL-6, TNFα, and IL-1β were measured using commercial ELISA kits (AbFrontier, Seoul, Korea) according to the manufacturer’s protocol.

Tissue samples were homogenized and solubilized in RIPA buffer (Sigma, St. Louis, MO). The lysates were centrifuged at 12,000 g for 10 minutes, and supernatants were collected. Lysates of 3T3-L1 adipocytes were prepared in a lysis buffer containing 25 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 25 mM sodium chloride, 10 mM sodium fluoride, 1 mM sodium vanadate, 1% Nonidet P-40 and protease inhibitor cocktails (Diagnostic Roche, Branchburg, NJ). Protein concentrations of lysates were detected with bicinchoninic acid method (Pierce, Rockford, IL). Equivalent proteins or same volume of conditioned media were loaded and separated on SDS-PAGE and then electro-transferred to nitrocellulose membranes. Membranes were incubated with anti-LCN2 (R&D System, Minneapolis, MN), anti-Phospho-NFκB p65 (Ser536) and anti-Phospho-STAT3 (Tyr705) (Cell Signaling, Danvers, MA) and anti-actin antibodies after blocking with TTBS (20 mM Tris-HCl pH 7.5, 0.5 M NaCl, 0.1% Tween-20) containing 5% milk (Fisher Scientific, Pittsburgh, PA). The membranes were then washed in TTBS and incubated with corresponding secondary antibodies conjugated to horseradish peroxidase (GE Healthcare Bio-Sciences Corp., Piscataway, NJ). The signals were detected by ECL plus Western Blotting Detection Reagents (GE Healthcare Bio-Sciences Corp., Piscataway, NJ).