FIG. 5.

NFATp binds to the −183 to −197 region within the TF promoter. (A) The sequences (5′ to 3′) of the oligonucleotides including the −183 to −197 region of the TF promoter (TF wt) and that of the TF mut are shown. Base pair substitutions incorporated in the TF mut are indicated by asterisks. The partially overlapping nucleotides corresponding to the κB-like and the NFAT sites are indicated. (B) Nuclear extracts from HUVECs pretreated or not with 200 ng of CsA per ml and then exposed to VEGF (50 ng/ml) were analyzed by EMSA with a probe spanning positions −183 to −197 of the TF promoter. A 30-fold molar excess of unlabeled TF −183 to −197, the corresponding oligonucleotide mutated at positions −186 to −194 (TF mut), or an NFAT consensus site of the IL-4 promoter oligonucleotides was added to the binding-reaction mixtures to detect the specific binding. Serological identification of the complexes was performed by addition of 0.5 μl of either preimmune serum (Preim.), anti-NFATp antiserum (67.1), or anti-NFAT family antiserum (796). Antisera and cold oligonucleotides were added to the binding-reaction mixture prior to addition of the probe.