EMSA of the sequence at the intron 6.

<p>A. Biotin-labeled general wild-type Sp1 oligonucleotide probe was incubated with increasing amounts (2, 4, and 8 µg) nuclear extracts from PBMCs, and then Sp1 DNA binding activity was detected by EMSA. B. Biotin-SP1 wild-type probe, consisting of a double-stranded oligonucleotide containing the DNA sequence spanning nts +5 to +34 of intron 6, was incubated with increasing amounts (2, 4, and 8 µg) nuclear extracts from PBMCs, and then Sp1 DNA binding activity were assayed by EMSA. C. Biotin-Sp1 mutant probe, consisting of a double-stranded oligonucleotide containing c.772+27G>C mutation in intron 6 and sequence spanning nts +5 to +34, was incubated with increasing amounts (2, 4, and 8 µg) nuclear extracts from PBMCs, and SP1 DNA binding activity was detected by EMSA. D. 8 µg of nuclear extracts were incubated with biotin-labeled Sp1 probe (lane 2), wild-type probe (lane 4), and mutant probe (lane 6) in the presence of 100-fold excess of unlabeled Sp1 probe (lane 3), unlabeled wild–type probe (designated as wt comp, lane 5), and unlabeled mutant probe (designated as mut comp, lane 7), then SP1 DNA binding activity was determined by EMSA.