Anti leptin

Total protein was isolated lysis buffer (Sigma-Aldrich). The protein lysates were separated by 8 to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes, which were then blocked in 5% bovine serum albumin and incubated overnight at 4 °C with the following primary antibodies: anti-phospho-PI3K p110α (1:1000, Cell Signaling Technology, Danvers, MA), anti-phospho-AKT (1:1000, Cell Signaling Technology), anti-phospho-mTOR (11,000, Abcam), anti-phospho-FAK (1:1000, Cell Signaling Technology), anti-PPAR-γ (1:500, Santa Cruz Biotechnology, Dallas, TX), anti-leptin (1:500, R&D systems), anti-TNF-α (1:500, Santa Cruz Biotechnology), and anti-GAPDH (1:3000, AbFrontier, Seoul, Republic of Korea). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA), and the bands were detected using an enhanced-chemiluminescence reagent (Bio-Rad).

RA-FLS, HUVEC, HSF and MH7A were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientifific, Waltham, MA, USA). THP-1 were cultured in RPMI1640 (Gibco) supplemented with 10% FBS. Cell lines were used from three to five passages in this experiment. All cells were cultured in medium supplemented with penicillin (50 U/mL) and streptomycin (50 μg/mL) and maintained in an incubator with 5% CO₂ at 37 °C.
FLS were cultured in DMEM medium supplemented with 5% (v/v) FBS, 5% healthy control serum and 5% RA serum (To avoid individual differences, the sera from 5 healthy controls or RA patients were combined into a sample.) after overnight serum starvation. Etomoxir (ETO, Sigma Adrich) was added into serum-treated RA-FLS. In order to block the effect of leptin, RA-FLS were handled by serum from HC or RA patients with or without human leptin antibody (anti-leptin, R&D Systems) and mouse IgG1 isotype control (R&D Systems). In some experiments, leptin-stimulated RA-FLS were handled in the presence or absence of ETO. And in other experiments, recombinant human leptin protein (leptin, R&D Systems)-stimulated RA-FLS was treated with or without AMPK inhibitor, compound C (CC, Cell Signaling Technology).