Analysis of expression of PAR1 and EPCR and cytoprotective signaling in PAR1-transfected HeLa cells. (A-C) The expression of PAR1 and EPCR in EA.hy926 cells (A) or HeLa cells (B) was evaluated by a cell-based ELISA or western blotting (C) using specific antibodies as described in “Materials and methods.” (D-H) Cell permeability in response to LPS (10 ng/mL) in HeLa cells transfected with PAR1 constructs. Confluent HeLa cells were transfected with the empty vector (D) or vector containing PAR-WT (F), PAR1-R46A (G), or PAR1-R41A (H), followed by treatment of cells with PAR1 agonists (20 nM APC, 2 nM thrombin alone, or 2 nM thrombin + 50 nM PC-S195A). Cells were then treated with LPS (10 ng/mL), and the permeability was monitored as described previously. (E) The efficiency of PAR1-WT transfection in HeLa cells was monitored by a cell-based ELISA. *P < .05 as compared with untreated control (UT) in panels A, B, and E; **P < .01 as compared with LPS in panels F, G, and H.