Figure 5.

Degradation of fibrinogen by CbiA-bound plasmin. CbiA (A), BBA70 (B), and BSA (C) (5 μg/ml each) were immobilized on microtiter plates, blocked with 0.2% BSA and incubated with plasminogen (10 μg/ml). Following several wash steps, a reaction mixture containing the plasminogen activator uPA (0.16 μg/ml) and fibrinogen (20 μg/ml) was added and plates were incubated at 37°C. Samples were taken at the indicated time intervals (hour in A,C, and minutes in B) and separated via Tris/Tricine SDS-PAGE. Upon transfer to nitrocellulose membranes, fibrinogen or its degradation products were detected in a Western blot analysis using a polyclonal anti-fibrinogen antibody. Control reactions included the lysine analog tranexamic acid (+T) and omission of plasminogen (–Plg) or uPA (–uPA). Reactions containing plasminogen, uPA and fibrinogen (PC), plasminogen and fibrinogen (NC) and fibrinogen alone (Fg) were also applied as additional controls. Shown are representative results from several independent experiments. The uncropped versions of panels (A-C) are presented in Supplementary Figure 2.