EPCR-mediated internalization of FVIIa and APC is dependent on dynamin and caveolar-mediated endocytosis. (A) CHO-EPCR were treated with a control vehicle (0.25% dimethyl sulfoxide) or Dynasore (80 μM), a specific inhibitor of dynamin GTPase, for 30 minutes. (B) CHO-EPCR cells were treated with control or K⁺-depleted buffer to inhibit clathrin-dependent endocytosis. (C) CHO-EPCR cells were treated with a control vehicle or mβCD (10 mM) for 30 minutes to disrupt caveolae. After aforementioned specific treatments, the cells were exposed to AF488-FVIIa (50 nM), AF488-APC (50 nM), or AF555-transferrin (Tfn; 300 nM) for 1 hour at 37°C. The fluorescence of internalized ligands was analyzed by confocal microscopy. The extent of internalization was quantified (D) by measuring the fluorescence intensity in the defined area corresponding to the recycling compartment. To identify the recycling compartment for the quantification, the cells were immunostained with EPCR mAbs (mean ± SEM, n = 15-25 cells).