Thrombin induces Egr-1 protein in primary endothelial cells.

HPAECs were grown on 6-well plates and starved overnight in EBM-2 medium containing 0.5% FBS. The cells were treated in the absence (C) or presence of 1.5 U/mL thrombin for the times indicated and subsequently harvested for whole-cell protein extracts as described in “Materials and methods.” In Western blot analysis, 20 μg of cell lysates was separated by 10% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with a polyclonal anti–Egr-1 antibody. The membranes were stripped and reprobed for β-actin to control for loading.