FIGURE 5.

Electrophoretic mobility shift assay demonstrating that cellular iron depletion using either DFO or 311 results in increased NF-Y DNA binding activity to the CCAAT-binding sequence present in the promoter region of GADD45α. A, MCF7 cells were incubated with either control medium, or this medium containing DFO (250 μm) or 311 (25 μm) for 24 h/37 °C. Nuclear protein extracts were then prepared and incubated with biotin-labeled oligonucleotide containing the CCAAT binding sequence found in the GADD45α promoter (WILD-TYPE target DNA; see “Experimental Procedures”) in the presence or absence of a 50-fold excess of unlabeled competitor WILD-TYPE target DNA. B, in parallel experiments, MCF7 cells were incubated as in A and nuclear lysates prepared and incubated with a biotin-labeled oligonucleotide containing a mutated CCAAT-binding sequence (MUTATED target DNA; see “Experimental Procedures”) in the presence or absence of a 50-fold excess of unlabeled competitor MUTATED target DNA. Results shown are typical gels from three experiments, while the densitometry is mean ± S.D. ***, p < 0.001, relative to the control.