Figure 5.

c-Jun and ATF-2 proteins specifically bind to the cyclin D1-CRE. (A) The ³²P-labeled DNA probe (≈25,000 cpm) was incubated for 30 min at room temperature with the in vitro-translated c-Jun, ATF-2 and ER proteins as indicated above each lane. For supershift assays, lysates were preincubated with the antisera for 15 min at room temperature before the addition of the radioactive probe. Open arrow indicates the ATF-2 homodimeric complexes and the closed arrow the ATF-2/c-Jun heterodimeric complexes. (B) Nuclear extracts (5 μg) of MCF-7 cells treated as described in Fig. 1A were incubated with 2 μg of poly dI-dC for 15 min at 4°C before the addition of the radioactive cyclin D1-CRE probe. For competition experiments, a 100-fold molar excess of unlabeled CRE or mutated CRE was mixed with the radioactive probe. (C) Nuclear extracts (5 μg) of MCF-7 cells treated with estradiol for 7 h were preincubated with 2 μg of poly dI-dC for 15 min at 4°C, then antisera were added for 15 min at room temperature and finally with radioactive probe. The arrow indicates the position of c-Jun/ATF-2-DNA complexes.