FIG. 3.

The Smad3-Smad4 binding site in the human c-Jun promoter is identified as a CAGA triplet located 3′ of the TATA box. (A) Schematic diagram of the −79 to +170 region of the c-Jun promoter. Four oligonucleotide sequences, named A through D, were designed to span the −18 to +170 region of the promoter. An additional oligonucleotide bearing a mutation in a CAGA triplet from +62 to +73 (BMUT) is also diagrammed (see text for additional discussion). The mutation changed the sequence from GACAGACAGACA to AGGAGCTTGCAA. (B) EMSA was performed by using the same −18 to +170 probe and HaCaT lysates as described for Fig. 2. A 100-fold molar excess of unlabeled oligonucleotides was incubated with the nuclear lysates before addition of radiolabeled probe, in order to compete with binding. The induced Smad3-Smad4 binding complex is indicated with an arrow. (C) EMSA was performed by using nuclear lysates from untreated mink lung cells or mink lung cells treated with TGF-β1 for 1 h and the same −18 to +170 probe. Radiolabeled probe was either the wild-type sequence from −18 to +170 or the mutated sequence from +62 to +73 (the CAGA triplet).