EMSA using oligonucleotide probes with RUNX1-binding sites 1A, 1B, 2, and 3 and nuclear extracts from PMA-treated HEL cells. (A) EMSA using wild-type oligonucleotide probe with sites 1A (−1498/−1493) and 1B (−1491/−1486), and nuclear extract from PMA-treated HEL cells. Lane 1 indicates probe alone; lane 2, probe with nuclear extract; lane 3, competition with excess unlabeled probe; lane 4, competition with unlabeled site 1A mutant probe; lane 5, competition with unlabeled site 1B mutant probe; lane 6, effect of RUNX1 antibody; lane 7, effect of nonspecific IgG; and lane 8, competition with unlabeled probe with sites 1A and 1B mutated. (B) EMSA using oligonucleotide probes with site 1A mutated (mutant probe 1A lanes 1-5) or site 1B mutated (mutant probe 1B lanes 6-10). Lanes 1 and 6 indicate probe alone; lanes 2 and 7, protein binding with nuclear extract; lanes 3 and 8, competition with excess respective unlabeled probe; lanes 4 and 9, effect of nonspecific IgG; and lanes 5 and 10, inhibition of binding with RUNX1 antibody. (C) EMSA using oligonucleotide probe with site 2 (−708/−703). Lane 1 indicates free probe; lane 2, protein binding with extract; lane 3, competition with excess unlabeled probe; lane 4, competition with unlabeled probe with site 2 mutated; lane 5, inhibition of binding with RUNX1 antibody; and lane 6, effect of nonspecific IgG. (D) EMSA using oligonucleotide probe with site 3 (−526/−521). Lane 1 indicates free probe; lane 2, protein binding with extract; lane 3, competition with excess unlabeled probe; lane 4, competition with unlabeled probe with site 3 mutated; lane 5, inhibition of binding with RUNX1 antibody: and lane 6, effect of nonspecific IgG.