Figure 3

Essential role of ERK activation in thrombin and carbachol-triggered upregulation of Egr-1 expression. (A) 39M1-81 cells were serum-starved for 24 hours and treated with thrombin (1 U/ml) or carbachol (100 μM) for 5 min. 39M1-81 cells were preincubated for 6 hours with PD98059 (50 μM) as indicated. Whole cell extracts were prepared and subjected to Western blot analysis. The blots were incubated with a monoclonal antibody directed against the phosphorylated active forms of ERK1 and ERK2. (B) SH-SY5Y neuroblastoma cells were incubated in medium containing 0.05% serum for 24 hours and treated with carbachol (100 μM) for 15 min. Whole cell extracts were prepared and subjected to Western blot analysis. The blots were incubated with a monoclonal antibody directed against the phosphorylated active forms of ERK1 and ERK2. (C) The activation of ERK is essential to connect thrombin and carbachol stimulation enhanced Egr-1 biosynthesis. 39M1-81 cells were preincubated for 6 hours with PD98059 (50 μM). Cells were stimulated with thrombin (1 U/ml) or carbachol (100 μM) for 1 hour. Nuclear extracts were prepared and subjected to Western blot analysis. The blots were incubated with an antibody directed against Egr-1.