Transcription driven by different 9E3/cCAF promoter constructs after thrombin stimulation.

(A) The long promoter of the 9E3/cCAF gene, −1503 to +32 bp (p1503; I), was subjected to 5′ deletions to obtain 3 additional promoter fragments as shown (II-IV). The transient transfections were performed as described in Figure 1C. p683 contains the elements necessary for basal stimulation as well as thrombin-stimulated transcription. The region between −1503 and −1116 bp may contain regulatory elements that repress gene activation by p1116, after thrombin stimulation. (B) The 5′ deletions of the p683 (II-V) were obtained by restriction enzyme digests and by PCR-based methods as described in “Materials and methods.” The transient transfections were performed as described in Figure 1C. After deletion of both Elk1 binding sites, a dramatic decrease in the response to thrombin occurred (p470, IV). The experiments shown in this figure are representative of several performed under the same conditions but with different batches of primary cells. Bars represent SEMs of 3 samples per condition in the same batch of cells.