MLN51 expression is upregulated in rheumatoid arthritis FLSs compared with osteoarthritis FLSs. (a) Total RNA sample (1 μg) was extracted from three rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs) and one osteoarthritis (OA) FLS with Trizol reagent. RT-PCR was performed with 5 ng of cDNA as a template and MLN51-specific or GAPDH-specific primers. The band for OA FLSs resulted from one (2–43) of the three OA FLSs. (b) Western blot analysis of MLN51 in FLS samples. RA FLSs (2–18, 2–36 and 2–38) and OA FLSs (2–43, 2–46 and 2–47) isolated from each patient were seeded at 5 × 104 cells per well in a six-well plate. FLSs grown in high-glucose DMEM supplemented with 10% FBS were harvested, separated by 10% SDS-PAGE, transferred to a nitrocellulose membrane and then proved with anti-hMLN51 rabbit serum (1:1,000 dilution) and horseradish peroxidase-conjugated anti-rabbit IgG (1:5,000 dilution). Data in (a) and (b) are representative of three or four separate experiments.