MEK1/ERK2 involvement in activation of 9E3/cCAF by thrombin.

(A) Immunoblot analysis of ERK2 phosphorylation. Extracts prepared from cells treated in the presence or absence of thrombin and in the presence of thrombin plus the MEK1 inhibitor PD98059 or the p38 inhibitor SB203580 were analyzed by immunoblot using antibodies to activated ERK2. Thrombin caused a high level of phosphorylation of ERK2; PD98059 dramatically decreased phosphorylation of ERK2 but SB203590 had virtually no effect. The same blot was stripped and reprobed with anti-ERK2 antibodies to show the levels of the ERK2 protein in each lane. (B) Immunoblot analysis with an antibody to the cCAF chemokine. Thrombin stimulated high expression of cCAF, whereas blocking phosphorylation of ERK2 via MEK1 inhibition by PD98059 inhibited the production of cCAF in cells treated with thrombin. (C) Dose-dependent effect of PD98059 on p683-induced activation of gene expression after stimulation by thrombin. Primary fibroblasts were transiently cotransfected as discussed in the Figure 2 legend. PD98059 lowered the stimulation of p683 by thrombin to levels comparable to those of the control in a dose-dependent manner.