Figure 1.

Stimulation of 39M1-81 fibroblasts with either thrombin or carbachol induces the biosynthesis of biologically active Egr-1. (A) 39M1-81 cells were serum-starved for twenty-four hours and then treated with thrombin (1 U/ml) or carbachol (100 μM) for 1, 3 or 6 hours as indicated and expression of Egr-1 was analyzed by immunoblotting. As a control, expression of the zinc finger protein Sp1 was analyzed. (B) Schematic representation of integrated provirus encoding an Egr-1 responsive luciferase reporter gene consisting of four binding sites for Egr-1 derived from the human synapsin I promoter, the human immunodeficiency virus TATA box, the adenovirus major late promoter initiator element and the luciferase open reading frame. (C) The newly synthesized Egr-1 protein is biologically active. 39M1-81 cells were infected with a recombinant lentivirus encoding the Egr-1 responsive reporter gene. The infected cells were stimulated with thrombin or carbachol for 16 hours. Cell extracts were prepared and analyzed for luciferase activities which were normalized to the protein concentrations. Each experiment illustrated here and in all subsequent figures was repeated a minimum of three times.