Interaction of NF-Y and USF1/2 in binding to the HOXB4 promoter through the HxRE-1 and HxRE-2 sites. (A) Note a slowly migrating band (arrow) detected when the intact HOXB4 core promoter (99 bp) was used as the probe in an EMSA with K652 cell nuclear extracts (NE) (lane 1). In lanes 2-4, 20 × molar excess of cold wild-type probe, of a truncated HOXB4 probe containing only HxRE-1, or of the truncated form containing the neutralizing HOXB4 mutation HxRE-1^* (as depicted in diagram below) was preincubated with K562 NE. In lanes 5 and 6, the HOXB4 core promoter containing either mutated HxRE-1^* or HxRE-2^* was used as the probe, in the absence of competing probe. (B) EMSA supershift, in which K562 NEs were pretreated with specific antibodies prior to being incubated with HOXB4 promoter probe. The upper EMSA band identified in lane 2 of panel A, which required both intact HxRE-1 and HxRE-2 sites, was disrupted by the antibody against either NF-Ya, USF1, or USF2, but not by the control IgG.