Northern blot analysis of Egr-1 and c-Fos induction in mouse endothelial cells. (top) Endothelial cells isolated from wild type, Par1–/–, and Par4–/– mice were grown to confluence, serum-starved overnight (12 hours), and stimulated with vehicle, the PAR4 agonist AYPGKF (500 μM), the PAR1 agonist TFLLRN (10 μM), or thrombin (10 nM) for 45 minutes. Total RNA was isolated and analyzed by Northern blot analysis. Hybridization for GAPDH mRNA was used to control for lane loading. This experiment was replicated 3 times. Quantitation of the results is shown (bottom) as the ratio of c-fos/GAPDH signal intensity normalized to control (mean ± SE; n = 3). Note the decreased response to thrombin in Par1–/– endothelial cells compared with wild-type and the similar magnitude or Egr-1 and c-Fos induction by thrombin and PAR4 agonist in Par1–/– cells.