Western blot analysis of hMLN51 in RA fibroblast-like synoviocytes (FLSs) treated with GM-CSF. Rheumatoid arthritis (RA) FLSs (2–18 and 2–38) isolated from the two patients with RA were seeded at 5 × 104 cells per well in a six-well plate. FLSs grown in high-glucose DMEM supplemented with 10% FBS were cultured further in the presence of granulocyte – macrophage colony-stimulating factor (GM-CSF) at 0, 50 and 100 ng/ml for 6 hours. Cells 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). This result is representative of three separate experiments.