<p>(A) Detection of longistatin bound on fibrin meshwork. Fibrinogen at different concentrations (3.75, 7.5, 15, 30 and 60 mM; final concentration) was mixed in the absence or presence of longistatin (10 µg) or an equal amount of t-PA or u-PA in a buffer (50 mM Tris–HCl, pH 7.5; 100 mM NaCl and 5 mM CaCl<sub>2</sub>) and thrombin (0.10 NIH unit/µl) was added immediately and was incubated at 25°C for 1 h. The clot was treated with anti-longistatin (1∶100), anti-t-PA (1∶100), anti-u-PA (1∶20) or pre-immune sera (1∶100). Bound antibodies were detected using green fluorescent-labeled secondary antibody (Alexa Flour 488 goat anti-mouse IgG). (B) Supernatant was analyzed by 12.5% SDS–PAGE under reducing conditions. (C) The target protein was extracted from the supernatant and its concentration was determined using micro-BCA reagent as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001312#s4" target="_blank">Materials and Methods</a>. The results are expressed as percentage of longistatin/t-PA/u-PA bound to the fibrin clot. Data represent mean ± SD, n = 3.</p
