FIG. 9.

GATA-2 is not included in RARα-RXRα-DNA complexes. 293T cells were transfected with an expression plasmid for either RARα, RXRα, or Flag-tagged GATA-2, separately, and the resultant nuclear extracts were mixed as indicated. Nuclear extract of the cells transfected with the empty vector was used to make the total amount of nuclear extracts the same. The mixtures of the nuclear extracts were then incubated with biotinylated oligonucleotides harboring RARE of either the DR5 (wild-type [wt] DR5; lanes 2 and 6) or the DR2 (wt DR2; lane 4) type, and the nucleotides were captured with streptavidin-agarose beads. The copurified proteins were then analyzed by Western blotting with anti-RARα, anti-RXRα, and anti-Flag antibodies. Nuclear extracts prior to mixing were analyzed as controls for appropriate expression of the proteins used (10% input; lane 1). Biotinylated oligonucleotides in which core recognition sites of the RARα-RXRα complex were mutated from GGTTCA and AGTTCA to GGTAGT and AGTAGT, respectively, were used as controls (mutant [mt] DR5 and DR2, lanes 3 and 5). Note that in no combination was GATA-2 copurified with RARα-RXRα-RARE complexes. The amounts of RARα and RXRα bound to DR5 are similar whether or not GATA-2 is included (lanes 2 and 6).