Thrombin and S1P synergistically induce Egr-1 mRNA expression and Egr-1 DNA binding activity. (A) Confluent monolayers of ECs were treated with or without 10 nM thrombin, 4 μM S1P, or 10 nM thrombin plus 4 μM S1P for 30, 60, and 90 minutes. The cells were then harvested and processed for determination of Egr-1 mRNA levels by semiquantitative RT-PCR. GAPDH mRNA levels were also measured as control. (B) Treated and untreated ECs for 30 minutes were subjected to EMSA using the Egr-1–specific probe.