The Elk1 transcription factor binds to the putative Elk1 binding elements of the 9E3/cCAF promoter and is activated by thrombin.

(A) EMSAs with extracts prepared from control chick embryo fibroblasts (CEFs), CEFs treated with thrombin, and CEFs overexpressing Elk1 and treated with thrombin. The conditions for the reactions are described in detail in “Material and methods.” All lanes have radiolabeled probe containing the Elk1 binding sequence (−493 to −483 bp) present in the promoter region of 9E3/cCAF. Extracts from untreated control cells 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). Incubation of the extracts from cells treated with thrombin, with the radiolabeled probe, caused a shift similar to that of untreated cells but much more pronounced (lane 7). In the presence of an antibody specific for the Elk1 protein (anti-Elk1) a supershift was detected both in the absence and presence of thrombin (lanes 4 and 8; arrow). However, this complex cannot be supershifted by an antibody specific to SAP-1 (lanes 5, 9, and 13), another member of the TCF transcription factor family, which has the ability to bind to the same element. When the extracts were treated with the antibody to activated Elk1 (anti-Ser383 phosphor-Elk1), the supershift was observed for the cells treated with thrombin only, indicating that this enzyme activates the Elk1 transcription factor. These same results were verified with cells overexpressing Elk1 where the shifts and supershifts are more obvious because the levels of the Elk1 transcription factor are much higher. (B) Immunoblot analysis of the activated Elk1 after treatment with thrombin. Anti-Ser383 phosphor-Elk1, the antibody that detects activated Elk1, showed that thrombin treatment increases the phosphorylation of this transcription factor. (C) Immunoblot analysis using the anti-Elk1 antibody that detects all forms of the Elk 1 transcription factor. The higher molecular weight band represents phosphorylated Elk1. (D) EMSA prepared with extracts of cells treated in the presence and absence of thrombin to verify the specificity of Elk1 shift and supershift complexes. To test the specificity of the shift we used a mutated Elk-1 binding oligonucleotide instead of wild type. To test the specificity of the supershift, we used excess unphosphorylated and phosphorylated C-terminal peptides of Elk-1 (GST-Elk1_310-428 and GST-P-Elk1_307-428, respectively) as competitors for the binding of the antibodies to Elk1 and a control antibody from the same company (Ser136 phosphor-Bad). (E) Immunoblot analysis of the phosphorylated and unphosphorylated peptides using both antibodies to the Elk1 C-terminal domain. The presence of 2 bands in the blot probed with anti-Elk1 is not surprising because it is common that during the purification of GST-fusion proteins partial cleavage of the protein occurs.