Influence of integrins and EGFR on FXII signalling. (A-D) Influence of integrin antibodies on FXII-induced ERK1/2 or Akt phosphorylation. (A) An antibody to phospho or total ERK1/2 was used; (C) an antibody to phospho (S473) and total Akt was used. In all panels the lane labeled “cells” is serum-starved HUVECs. (A) In lanes 2 and 3, HUVECs were incubated with 62nM FXII plus 50μM Zn²⁺ alone or in the presence of 100μM PD98059. In lanes 4-8, cells were incubated with 62nM FXII plus 50μM Zn²⁺ in the presence of 188nM ASC-1, HA5, 6S6, or P4C10 or 63nM AIIB2, respectively. The integrin the antibody is directed toward is indicated on the figure in parentheses; Mab AIIB2 is directed to β₁ integrin. (A) The space in the immunoblot indicates nonconsecutive lanes of the same gel. (B) A graphic representation of 3 or more experiments (means ± SD) shown in panel A. (C) Scan of nonconsecutive lanes on the same immunoblot. In lanes 2 and 3, HUVECs were incubated with 62nM FXII plus 50μM Zn²⁺ alone or in the presence of 30nM Wortmannin. In lanes 4-8, cells were incubated with 62nM FXII plus 50μM Zn²⁺ in the presence of 188nM Mabs 6S6, P4C10, P1D6, ASC-1, or 63nM AIIB2, respectively. (D) Graphic representation of 3 or more experiments (means ± SDs) in panel C. (B,D) An asterisk over a bar graph indicates P ≤ .05. (E-F) Influence of peptides to the uPAR-integrin interaction sites or an EGFR inhibitor on FXII-induced phosphorylation of ERK1/2 or Akt. (E) Serum-starved HUVECs were treated with 62nM FXII and 50μM Zn²⁺ alone or in the absence or presence of 50μM PD98059, Wortmannin, 50nM AG1478, 300μM peptide NLD9 or peptide IQE13, respectively, and examined for ERK1/2 or Akt phosphorylation. (E) Representative experiment; (F) bar graphs of the means ± SDs of 3 or more experiments of panel E. (F) An asterisk over a bar graph indicates P ≤ .05 compared with FXII-treated HUVECs alone.