Mutations in Sp1 sites interfere with DNA-protein complex formation. Gel mobility shift assays were performed using 32P-end–labeled core promoter fragment (bp −30 to −92) and 1 μg of nuclear extracts from uninduced (A) or 4-day induced (B) K562 cells. Two protein complexes were formed, as indicated by the arrows. Unlabeled double-stranded DNA fragments consisting of either the 35-bp fragment containing intact Sp1 binding sites (bp −30 to −65) or the 35-bp fragments containing the 5′ mutated Sp1 binding site (Δ 5′ Sp1), the 3′ mutated Sp1 binding site (Δ 3′ Sp1), or mutations of both Sp1 binding sites (Δ dbl Sp1) or an irrelevant 119-bp double-stranded DNA fragment were used as competitive inhibitors. The molar excess of the unlabeled competitor is indicated.
