Fig. 5.

AP4 binds to the E-box motif of the DBH upstream promoter. Oligonucleotide DBH/AP4 containing the E-box (CANNTG) motif of the DBH promoter was radiolabeled and used as a probe. (a) Two μl of the in vitro translated AP4 proteins were incubated with the radiolabeled probe (lane 2) and 0.2 μg of AP4 antibody was added to the reaction (lane 3). For competition, 50-fold (lanes 4, 7, 10), 200-fold (lanes 5, 8, 11), or 1,000-fold (lanes 6, 9, 12) molar excess of unlabeled oligonucleotides DBH/AP4 (lanes 4-6), Sp1 (lanes 7-9), and SV40/AP4 (10-12) were added to the reaction mixture before the addition of radiolabeled probe. The same amount of in vitro translated protein using an empty vector was incubated with the probe and did not generate any complex (lane 1). (b, c) The ³²P-labeled DBH/AP4 probe was incubated with 3 μg of HeLa (b) or 1 μg of SK-N-BE(2)C (c) nuclear extracts (lane 2, 9). For competition, 50-fold (lanes 3, 6), 200-fold (lanes 4, 7), or 1,000-fold (lanes 5, 8) molar excess of unlabeled oligonucleotides DBH/AP4 (lanes 3-5) and Sp1 (lanes 6-8) were added to the reaction mixture before the addition of radiolabeled probe. AP4 specific antibody was incubated with the probe and nuclear extracts (lane 10). In lane 1, the probe was incubated without nuclear extracts. (d) The ³²P-labeled DBH/AP4 probe was incubated with 2 μl of in vitro translated AP4 proteins (lane 2). For competition, 50-fold (lanes 3, 6), 200-fold (lanes 4, 7), or 1,000-fold (lanes 5, 8) molar excess of unlabeled oligonucleotide wild type DBH/AP4 (lanes 3-5) and mutant type DBH/AP4 (lanes 6-8) were used. The same amounts of in vitro translated proteins using an empty vector were incubated with the probe and did not generate any complex (lane 1). Specific protein-DNA complex and supershifted complex were indicated by an arrow head and an arrow, respectively.