The two Sp1 binding sites are required for α₂ integrin gene regulation in other models of megakaryocytic differentiation. (A) The relative activities of the intact α₂ integrin core promoter construct and the Sp1 mutant constructs were determined in the megakaryoblastic leukemia cell, Dami. The promoter activity of the original construct pα₂92-CAT, the mutant construct pα₂Δ5′ Sp1-CAT, pα₂Δ3′ Sp1-CAT, pα₂Δdbl Sp1-CAT, and pCAT-Basic was compared with the pα₂ 65-CAT construct after transient transfection into Dami cells. Cotransfection with pRSV-luciferase was used to control for transfection efficiency. After 48 hours of incubation, cell extracts were assayed. After normalization for transfection efficiency, CAT activity of the constructs was determined by thin-layer chromatography and differential extraction. The average and standard deviation of the CAT activity of the mutant constructs relative to the pα₂65-CAT construct was determined. (B) Nuclear extracts from Dami cells bind the α₂ integrin core promoter. The 62-bp fragment of the α₂ integrin promoter (extending from bp −30 to −92) specifically bound nuclear proteins from Dami cells producing two DNA-protein complexes. Gel mobility shift analysis was performed with the ³²P-end–labeled double-stranded DNA fragment containing bp −30 to −92 and 1 μg of nuclear protein from Dami cell lines. Two DNA-protein complexes are indicated with 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 fragment containing the 5′ mutated Sp1 binding site (Δ5′ Sp1), the 3′ mutated Sp1 binding site (Δ3′ Sp1), or mutations of both Sp1 binding sites (Δdbl Sp1) or an irrelevant 119-bp double-stranded DNA fragment were used as competitive inhibitors. The molar excess of the unlabeled competitor is indicated.