Ferritin and HKa-Mediated Signaling Figure 7. Ferritin blockes HKa-mediated disruption of the complex between uPAR and integrin a5b1. HUVEC cells were allowed to adhere onto vitronectin-coated dishes for 2 hours in the presence of HKa (50 nM), ferritin (100 nM), or the combination of HKa plus ferritin. A. Cells were lysed, immunoprecipitated with anti- a5b1 antibody, and the immunoprecipitates analyzed by western blotting with anti-uPAR and anti-b1 antibodies. B. Bands were quantified by densitometry. Shown are means and standard deviation of 3 independent experiments. C. 2 mg of recombinant proteins were electrophoresed on a SDS-PAGE. Binding to ferritin was analyzed by ligand blotting. D. Model of ferritin effects on HKa signaling in endothelial cells. doi:10.1371/journal.pone.0040030.g007 factor (bFGF; BD Biosciences) and 10 mM ZnCl2 in M199 basal starvation medium (GIBCO) with the following reagents: two chain high molecular weight kininogen (HKa; Enzyme Research Laboratories); HGK-rich peptide 474DHGHKHKHGHGHGKHKNKGKKNGKHNGWK502(A- naspec); control peptide 440GHGLGHGHEQQHGL- GHEHKFKLDDDLEHQGGHVL473(Anaspec); spleen ferritin (Scripps Laboratories), recombinant HFt or LFt (below) for 24 hours prior to MTT assay. For clonogenic assays, colonies were allowed to grow for 10 days before fixing and staining with crystal violet. Effect of HKa on signaling molecules in HUVECs Cells were seeded at 46105 cells per/well in a 6- well plate in growth medium and allowed to adhere for 4 hours before the medium was replaced with M199 basal medium without growth factors. Following overnight incubation, the cells were either left untreated or treated with HKa, HGK-rich peptide, control peptide or 100 mM PD98059 (Promega) in the presence or absence of ferritin in medium containing 20 ng/ml of bFGF or 62 nM FXII (Haematologic Technologies Inc) and 10 mM ZnCl2 for 24 hours. Cells were lysed in Triton X-100 (TX-100) lysis buffer (50 mM Tris, pH 7.5, 150 mM sodium chloride, 0.5% TX- 100) supplemented with protease and phosphatase inhibitor cocktails (Roche) and protein concentration of the clarified samples determined using BCA protein assay kit (Pierce). Proteins were separated by SDS PAGE and transferred into a polyviny- lidene difluoride (PDVF) membrane. The membranes were blocked in 5% BSA in Tris-buffered-saline containing 0.05% Tween-20, and probed with the following antibodies: anti- phospho-MAPK 44/42, anti-total-MAPK 44/42, anti-phospho- Paxillin Y118, anti-Paxillin, anti- phospho-FAK Y397, anti-FAK, anti-phospho-Akt (Akt S473) (Cell Signaling); anti-GAPDH (Fitzgerald Industries International, Inc.); Streptavidin-HRP (Pierce); and anti-GST (Sigma). Secondary antibodies were either horseradish peroxidase (HRP)-conjugated goat anti-rabbit immu- noglobulin G (IgG) or HRP-conjugated goat anti-mouse IgG (Biorad). Signals were detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and membranes PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e40030