Sp1 functions at the −70 position of the β₃ promoter. (A) Sp1 mutational analysis. HEL cells were transfected with equivalent amounts of −146 wild-type and Sp1 mutant constructs, assayed for luciferase activity, and expressed as fold activation over background. The mutant construct contained the 2-bp substitution in probe mMS7 that destroyed the Sp1 site at position −70. Each experiment was performed five times. (B) Cotransfection of Sp1 enhances expression. HEL cells were transfected with the indicated plasmids and assayed for luciferase activity. Each experiment was performed four times. Twenty micrograms of −146 wild-type or −146 mPw1mut and 4 μg of CMV-Sp1 or CMV-empty (total plasmid DNA, 24 μg) were transfected in each experiment.