Figure 6

Thrombin-regulated expression of Egr-1 requires the activation of PKC. 39M1-81 cells (A) or 1321N1 astrocytoma cells (C) were preincubated with TPA (100 ng/ml) or vehicle for twenty-four hours and then stimulated with either thrombin (1 U/ml) or carbachol (100 μM) for 1 hour. Nuclear extracts were prepared and subjected to Western blot analysis using an antibody directed against Egr-1. As a control, cells were stimulated with EGF (10 ng/ml) in the presence or absence of TPA (A, lower panel). (B) 39M1-81 cells were preincubated with the PKC inhibitor GF109203X (10 μM) or vehicle for 30 min and then stimulated with either thrombin (1 U/ml) or carbachol (100 μM) for 1 hour. Nuclear extracts were prepared and subjected to Western blot analysis using an antibody directed against Egr-1. (D) 39M1-81 cells were preincubated with TPA (100 ng/ml) or vehicle for twenty-four hours and then stimulated with either thrombin (1 U/ml) or carbachol (100 μM) for 5 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.