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Fig. 3. Analysis of TGF-ß activation in culture supernatants of ECs and SMCs by Plg fragments. (A) K1-3 and mini-Plg purity was analysed by Coomassie stain (CB) and by western blotting using the K1-3-specific mAb 2PG or the mini-Plg-specific mAb 4PG (top). The plasmin activity in the preparations was analysed using the plasmin-specific colorimetric substrate S-2251 before and after activation with active uPA (bottom). HUVECs (B) or HUVSMCs (C) were cultivated in 96-well plates (5000 cells per well) and treated with 240 nM Plg and 60 nM, 120 nM or 240 nM mini-Plg (B), or with 240 nM mini-Plg (C) in assay medium for 12 hours. For analysis of total TGF-ß (LTGF-ß plus active TGF-ß), culture supernatants were heated for 10 minutes at 80°C. For analysis of active TGF-ß alone, untreated supernatants were used. Active or total TGF-ß was then analysed by the MLEC assay.
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