|Western blot analysis|
FLSs cultured (2.5 × 105 cells) in 60-mm dishes were serum-starved overnight and stimulated by adiponectin (1 or 10 μg/mL) or IL-1β (0.1 or 1 ng/mL) for 24 hours. The cells were subsequently washed twice in phosphate-buffered saline and treated with 50 μL of lysis buffer (20 mM Tris-Cl [pH 8.0], 150 mM NaCl, 1 mM EDTA [ethylenediaminetetraacetic acid], 1% Triton X-100, 20 μg/mL chymostatin, 2 mM PMSF [phenylmethylsulphonyl fluoride], 10 μM leupeptin, and 1 mM AEBSF [4-(2-aminoethyl)benzenesulfonyl fluoride]). The samples were separated using 12% SDS-PAGE and were then transferred to Hybond-ECL [enhanced chemiluminescence] membranes (Amersham, now part of GE Healthcare, Little Chalfont, Buckinghamshire, UK). The membranes were first blocked with 6% nonfat milk dissolved in TBST buffer (10 mM Tris-Cl [pH 8.0], 150 mM NaCl, 0.05% Tween 20). The blots were then probed with various rabbit polyclonal antibodies for COX-2 and β-actin (Cell Signaling Technology, Inc., Danvers, MA, USA) diluted 1:1,000 in Tris-buffered saline at 4°C overnight and incubated with 1:1,000 dilutions of goat anti-rabbit IgG secondary antibody coupled with horseradish peroxidase. The blots were developed using the ECL method (GE Healthcare). For re-probing, the blots were incubated in the stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl [pH 6.7]) at 50°C for 30 minutes with occasional agitation.