Fig. 7.
Fig. 7. Elk1 activation by thrombin occurs via MEK1/ERK2. / (A) EMSA with extracts prepared from cells overexpressing Elk1 and treated with thrombin in the absence and presence of the MEK1 inhibitor PD98059. The conditions for the reactions are described in “Material and methods.” All lanes have radiolabeled probe containing the Elk1 binding sequence present in the promoter region of 9E3/cCAF (−493 to −483 bp). Extracts from cells treated with thrombin contained factors that caused shift complexes to occur (lane 2, arrowheads). These complexes did not form when the incubation with the radiolabeled probe was done in the presence of 25-fold excess of unlabeled probe (lane 3). Extracts from cells treated with thrombin in the presence of PD98059 contained reduced amounts of factors but caused similar, albeit less pronounced, shift complexes (lane 6, arrowhead). Incubation of the extracts from lanes 2 and 6 with the radiolabeled probe in the presence of an antibody specific for the Elk1 protein (anti-Elk1) resulted in a supershift (lanes 4 and 8, arrow). However, when the extracts from lanes 2 and 6 were incubated with an antibody to the activated Elk1 (anti-Ser383 phosphor-Elk1) only the extracts from cells treated with thrombin in the absence of PD98059 resulted in a supershift (compare lanes 5 and 9). (B) Immunoblot analysis of activated Elk1 detected with anti-Ser383 phosphor-Elk1 in the presence of PD98059 or SB203580. The latter inhibitor slightly decreases thrombin-induced phosphorylation of Elk1, whereas the former prevented phosphorylation. (C) Effect of MEK1 on activation of the Elk1 transcription factor. The same heterologous expression system used in Figure 5 was used for these experiments, and the conditions for the transfections were described in the Figure 2legend. When appropriate, the pcDNA 3.1 plasmid was used to maintain the total amount of DNA transfected at 6 μg. When cells were cotransfected with 2 μg of pFR-Luc and 1 μg of the fusion protein plasmid pFA-Gal4dbd-Elk1AD, no significant activation of the reporter was observed (iii), but cotransfection of the same amounts of pFR-Luc and pFA-Gal4dbd-Elk1AD in conjunction with 1 μg of the plasmid for MEK1 overexpression, resulted in a significant activation of the reporter gene (iv). This activation was inhibited by PD98059 (v). As a negative control, transfection with pFR-Luc alone (2 μg) or cotransfections of the Gal4 DNA binding domain (Gal4dbd; 1 μg) and pFR-Luc (2 μg) were performed. These experiments were performed several times with different batches of primary cells. Here we show representative experiments. Bars represent SEMs of 3 samples/condition in the same batch of cells.

Elk1 activation by thrombin occurs via MEK1/ERK2.

(A) EMSA with extracts prepared from cells overexpressing Elk1 and treated with thrombin in the absence and presence of the MEK1 inhibitor PD98059. The conditions for the reactions are described in “Material and methods.” All lanes have radiolabeled probe containing the Elk1 binding sequence present in the promoter region of 9E3/cCAF (−493 to −483 bp). Extracts from cells treated with thrombin contained factors that caused shift complexes to occur (lane 2, arrowheads). These complexes did not form when the incubation with the radiolabeled probe was done in the presence of 25-fold excess of unlabeled probe (lane 3). Extracts from cells treated with thrombin in the presence of PD98059 contained reduced amounts of factors but caused similar, albeit less pronounced, shift complexes (lane 6, arrowhead). Incubation of the extracts from lanes 2 and 6 with the radiolabeled probe in the presence of an antibody specific for the Elk1 protein (anti-Elk1) resulted in a supershift (lanes 4 and 8, arrow). However, when the extracts from lanes 2 and 6 were incubated with an antibody to the activated Elk1 (anti-Ser383 phosphor-Elk1) only the extracts from cells treated with thrombin in the absence of PD98059 resulted in a supershift (compare lanes 5 and 9). (B) Immunoblot analysis of activated Elk1 detected with anti-Ser383 phosphor-Elk1 in the presence of PD98059 or SB203580. The latter inhibitor slightly decreases thrombin-induced phosphorylation of Elk1, whereas the former prevented phosphorylation. (C) Effect of MEK1 on activation of the Elk1 transcription factor. The same heterologous expression system used in Figure 5 was used for these experiments, and the conditions for the transfections were described in the Figure 2legend. When appropriate, the pcDNA 3.1 plasmid was used to maintain the total amount of DNA transfected at 6 μg. When cells were cotransfected with 2 μg of pFR-Luc and 1 μg of the fusion protein plasmid pFA-Gal4dbd-Elk1AD, no significant activation of the reporter was observed (iii), but cotransfection of the same amounts of pFR-Luc and pFA-Gal4dbd-Elk1AD in conjunction with 1 μg of the plasmid for MEK1 overexpression, resulted in a significant activation of the reporter gene (iv). This activation was inhibited by PD98059 (v). As a negative control, transfection with pFR-Luc alone (2 μg) or cotransfections of the Gal4 DNA binding domain (Gal4dbd; 1 μg) and pFR-Luc (2 μg) were performed. These experiments were performed several times with different batches of primary cells. Here we show representative experiments. Bars represent SEMs of 3 samples/condition in the same batch of cells.

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