Fig. 4. The NF-B-binding site of the uPAR promoter is required for ß₃-endonexin-mediated downregulation of the uPAR gene transcription. (A) CAT-assay. CHO cells were transiently co-transfected with either the uPAR promoter construct (uPAR-CAT -398) described in Fig. 2A or a CAT-reporter construct driven by the -398 by uPAR promoter sequence mutated at the NF-B motif at -45 bp (uPAR-CAT mt-45). Additionally, a luciferase vector for normalization, and 3 µg of expression vectors encoding either the short or long form of ß₃-endonexin or the mock expression vector (GFP) were transfected. The results are representative of three independent experiments. (B) EMSA. Recombinant GST-fusion proteins, GST/En-S and GST/En-L, or GST control were added in increasing ratios (1:2, 1:3, 1:4) to nuclear extracts of IL-1ß-activated HUVECs. Binding of B oligonucleotides was evaluated by EMSA.