Extracellular matrix and pericellular proteolysis.
(A) Western blot analysis of fibrinogen and proteolytic fragments in conditioned media of VG and N. Human fibrinogen (100 µg/mL) was incubated with saline (0), plasmin (Pn, 500 nM and 100 nM) and elastase (El, 100 nM and 20 nM) for 2 h at 37°C, to generate reference fibrinogen degradation products (FDPs). Pairs of conditioned media from 4 patients were analyzed under conditions of reduced disulfide bonds using a rabbit anti-human fibrinogen antibody, which recognizes both the intact Aα, Bβ, and γ chains, γ-γ dimers, and FDPs (positions indicated by brackets on the right-hand side). (B) Western blot analysis of fibronectin and its proteolytic fragments in pairs of conditioned media from 5 patients using a polyclonal anti-fibronectin antibody. Purified fibronectin (0.5 µg per well) was used as a control. (C) Western blot analysis of shedding of soluble species of uPAR in pairs of conditioned media from 5 patients, using a mouse monoclonal anti-uPAR D2 domain antibody. Purified recombinant human uPAR (15 ng per well) was used as a reference, and the position of the intact three-domain (D1D2D3) and truncated two-domain (D2D3) species under reducing conditions is indicated on the right. For all Western blots, positions in the gel of molecular mass standard proteins used for calibration are indicated on the left.