FIGURE 4 .

After thrombin treatment (2 U/mL) for 2 hours, A549 cells were harvested by centrifugation (1000 rpm for 5 minutes). Cytosolic and membrane fractions were isolated from the lysed cells using a Mitochondrial/Cytosol Fractionation Kit. Cell lysates were prepared, and then immunoblotted with antibodies for PKCα, PKCδ, and PKCε. Experiments were performed three times, and representative immunoblots are presented. Thrombin increased the migration of PKCα, PKCδ, and PKCε from cytosol to membrane fractions. PAR-1 siRNA or pretreatment with argatroban inhibited thrombin-induced PKCα, PKCδ, and PKCε activation. Addition of GÖ6976 (10 nM) to thrombin inhibited migration of PKCα; addition of rottlerin (4 μM)-inhibited migration of PKCδ; PKCε antagonist peptide (10 μM) inhibited migration of PKCε.