Mouse lcn2

After treatments, total cell lysates were obtained using lysis buffer (10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 0.5% Triton X-100, 1 mM PMSF), freshly supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA), and then centrifuged at 14,000 g for 20 min. SDS-PAGE and blotting procedures were performed as previously described [28] . Immunoblots were incubated with the specific antibody against (NOS-2 or COX-2 (Cell Signaling, Danvers, MA, USA), IL-6 or IL-8 (Santa Cruz Biotechnology, Dallas, TX, USA), MMP13 (Abcam, Cambridge, UK), mouse LCN2, or human LCN2 (R&D Systems, Minneapolis, MN, USA)).
The immune complexes were detected using anti-rabbit (GE Healthcare, Chicago, IL, USA), anti-mouse (both from GE Healthcare, Chicago, IL, USA) or anti-goat (Santa Cruz Biotechnology, Dallas, TX, USA) horseradish-peroxidaselabeled secondary antibodies and Immobilon ECL. To verify equivalent protein loading, the membranes were incubated with anti-GAPDH antibody (Sigma-Aldrich).
The signals generated were detected in ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc.) and analyzed with Image Lab 6.0.1 Software (Bio-Rad Laboratories, Inc., Hercules CA, USA).