Fig. 1. VEGFR-1 is a component of the extracellular matrix deposited by EC. (A) HUVEC or normal human fibroblasts (Fb) were seeded into 96-multiwell culture plates and incubated for 72 hours. Cells were mechanically detached and the amount of VEGFR-1 or fibronectin (FN) deposited in each well was evaluated by ELISA using anti-VEGFR-1 or anti-fibronectin antibodies. Histograms represent the mean absorbance value of medium from triplicate wells ± s.e.m. The experiment was repeated at least three times with comparable results. (B) Extracellular matrix deposited by HUVEC was immunostained after cell detachment with an anti-sVEGFR-1 antibody or with an anti-fibronectin antibody. Both sVEGFR-1 and fibronectin were detectable in the matrix deposited by the EC. Scale bar: 5 µm. (C,D) Immunoblotting analysis of VEGFR-1 polypeptides in HUVEC extracts and the ECM produced by these cells, using antibodies recognizing (B) the extracellular (AF327) or (C) the intracellular (C-17) region of VEGFR-1. Equal volumes of cell extract, ECM material or total samples (in which cells and ECM were collected together), prepared as described in Materials and Methods, were analysed. Upper panels show the immunoblot analysis of the samples using the anti-human fibronectin antibody FN-15. The molecular weight markers in kDa are indicated in C.