Evidence for selective PAR1 and PAR2 silencing in PAR1 or PAR2 siRNA–transfected cells. Fibroblasts were transfected with PAR1 or PAR2 siRNA as described in “Materials and methods.” (A) Sixty hours after the transfection, the cells were removed from the dish using EDTA solution (no trypsin), and the cell suspension was divided into 4 aliquots, and stained with TF mAB (TF9H10, 10 μg/mL), PAR1 mAB (ATAP2, 10 μg/mL), anti–rabbit PAR2 IgG (100 μg/mL), and appropriate control IgGs. The stained cells were subjected to FACS analysis. (B) Mock- and siRNA-transfected cells cultured in glass-chambered slides were loaded with Fluo-4 AM as described in “Materials and methods” and mounted on a microscope stage. After obtaining live fluorescence images for 30 to 45 seconds, control vehicle, or PAR1 or PAR2 agonist peptide (25 μM) was added to the cells and the imaging was continued for 5 minutes. The plots represent intracellular Ca²⁺ levels.