Effect of negative regulatory elements in the vWF gene promoter on the transcriptional activity of the TK promoter. Blunt-ended fragments of the vWF gene 5′-flanking sequence were ligated in the sense or antisense direction into the BamHI site of pBLCAT2 upstream of the TK promoter. Transfections were performed in HUVECs and HeLa cells as described in the Materials and Methods. Forty-eight hours after transfection, cell extracts were prepared and aliquots, normalized for transfection efficiency, were used for the determination of CAT activity. The inserts are schematized on the left. The CAT activity of the vector pBLCAT2 with the TK promoter alone is set at 100%. The CAT activities associated with the other vectors are expressed relative to the activity of pBLCAT2 and are given on the right. (▪) HUVECs; () HeLa cells. The results are the mean ± SD of 4 to 6 experiments.