Figure 3.

Confirmation that NF-Y binds to the CCAAT sequence. Nuclear extracts of HepG2 cells were prepared according to the procedure of Attardi and Tjian (34, 36). The ³²P-labeled DNA probe (nucleotides −92 to −40) was generated by using the Klenow fragment of DNA polymerase I. All other experimental conditions were as described (34) and as in Materials and Methods. Lane 1, DNA probe only; lane 2, DNA probe incubated with cell extract; lane 3, same as in lane 2, except that the DNA–protein complexes were incubated with anti-NF-Ya antibodies; lane 4, as in lane 3, but the binding reaction contained a 50-fold excess of the unlabeled DNA probe; lane 5, as in lane 4, except that the unlabeled DNA probe contained an additional 4 bp that were inserted between the NF-Y- and Sp-1-binding sites; lane 6, as in lane 4, except that the NF-Y-binding site in the unlabeled DNA probe was mutated.