Soluble il 6r

Protein extracts were prepared from p19-expressing HUVECs, which were untreated or treated with human IL-6 (200 to 1000 ng/ml; R&D Systems) and soluble IL-6R (200 to 2500 ng/ml; R&D Systems), and from human gp130-expressing BAF-130 cells, which were untreated or treated with human IL-6 (200 to 1000 ng/ml), soluble IL-6R (200 to 2500 ng/ml), or p19 protein (1 μg/ml; Abnova), in lysis buffer containing 10 mM tris-HCl (pH 7.5), 150 mM NaCl, 0.2% NP-40 (Abcam), 1 mM NaF, 1 mM EDTA, 10% glycerol, complete protease inhibitor cocktail (Roche), 2 mM sodium orthovanadate (Sigma-Aldrich), pepstatin A (1 mg/ml; Sigma-Aldrich), leupeptin hemisulfate salt (1 mg/ml; Sigma-Aldrich), and aprotinin (1 μg/ml; Sigma-Aldrich). Rabbit polyclonal IgG against human gp130 (Millipore) and control rabbit polyclonal IgG (Jackson ImmunoResearch Laboratories) were individually incubated (4 μg) with washed protein G Dynabeads (50 μl; Life Technologies) and incubated for 10 min at room temperature. After magnetic separation, the antibody-bead complex was added to the cell lysates (500 mg) followed by incubation for 18 hours at 4°C with rotation. Bead-antibody-protein complexes were recovered by magnetic separation. Target antigen was eluted by heat treatment (for 10 min at 70°C).