Recognition of PARs by immunoblot and cell surface binding. (A) Immunoblotting of platelet and PAR-transfected COS7 cell lysates with PAR antisera. COS7 cells were transfected with plasmids directing expression of mouse PAR1 or PAR3 or with empty vector. Transfected COS7 lysates and lysates of washed mouse platelets were analyzed by SDS-PAGE and immunoblot with PAR1, PAR3, or preimmune IgG (see the Materials and Methods). Preincubation of the PAR1 or PAR3 IgG with the peptide antigen to which each was raised (final peptide concentration, 3.3 nmol/L; overnight at 4°C) ablated their ability to recognize PAR1 or PAR3, respectively (not shown). This experiment was replicated four times. (B) Selective recognition of PARs on the COS7 cell surface. COS7 cells transfected with mPAR1 or mPAR3 expression vectors or with empty vector were fixed briefly with 2% paraformaldehyde, incubated with PAR1 or PAR3 IgG (60 μg/mL) as indicated, and then washed extensively. Antibody bound to the surface of unpermeabilized cells was detected using horseradish peroxidase-conjugated second antibody by cell surface ELISA.17 Data shown are the mean ± SEM (n = 4); binding to empty expression vector-transfected cells (0.14 OD units for PAR3 IgG; 0.08 for PAR1 IgG) was subtracted. This experiment was replicated twice.