Oligomerization of the αC domains amplifies adhesion-dependent activation of FAK and ERK. HUVECs were either kept in suspension or plated in serum-free DMEM for 2 hours on tissue culture plates coated with 20 μg/mL αC monomers or αC(FXIII) oligomers. (A) Phosphorylation of FAK tyrosines 397, 577, and 861 was examined by SDS-PAGE and immunoblotting with specific polyclonal antibodies (see “Materials and methods”). Overall tyrosine phosphorylation of FAK was tested by immunoprecipitation of FAK, followed by SDS-PAGE and immunoblotting with polyclonal antiphosphotyrosine antibodies. (B) Adhesion-dependent phosphorylation of ERK was analyzed by immunoblotting with antibodies against dually phosphorylated ERK1/2 and total ERK1/2. Panels A and B are representative of 3 independent experiments for FAK and ERK.