KLK12 neutralization modifies fibronectin matrix assembly by endothelial cells and fibroblasts. (A) Description of the protocol used to generate pAbFN-KLK12, an antibody that recognizes the KLK12 cleavage site. (B) Test of pAbFN-KLK12′s specificity on KLK12-generated FN fragments. KLK12-generated proteolytic fragments of FN and FN-EDB were prepared as in Fig. 2A and then analyzed in Western blot. (C and D) The impact of KLK12 neutralization on FN matrix assembly by ECs (C) and fibroblasts (D), using a neutralizing antibody that targeted the KLK12 cleavage site on FN (pAbFN-KLK12). Human ECs were treated with 10 nM KLK12 for 24 h in basal medium with or without FN (500 nM) and pAbFN-KLK12 or the isotype control (500 nM). Fibroblasts were treated with 10 nM KLK12 for 24 h in basal medium with or without pAbFN-KLK12 (50 nM and 500 nM). After treatment, cells and matrix were fixed, the FN fibrils were immunodetected (monoclonal anti-FN antibody IST-2, green channel) and DNA was stained using Hoechst reagent (blue channel). Scale bar = 200 µm.