FIG. 5.

RXRα inhibits recruitment of RARα to GATA-2-GATA motif DNA complex. 293T cells were transfected with an expression plasmid for either Flag-tagged GATA-2, RARα, or RXRα, separately. The resultant nuclear extracts were then mixed as indicated. Nuclear extract of the cells transfected with an empty vector was used to make the total amounts of nuclear proteins equal. The nuclear extracts containing the indicated proteins were then incubated with biotinylated oligonucleotides harboring GATA motifs (wild-type [wt] GATA), and the nucleotides were captured by streptavidin-agarose beads. The resultant copurified proteins were analyzed by Western blotting with anti-Flag, anti-RARα, and anti-RXRα antibodies. Nuclear extracts prior to mixing were analyzed as controls for appropriate expression of the proteins used (input; 10%). Biotinylated oligonucleotides in which GATA core recognition motifs were mutated to TTTA (mutant [mt] GATA) were used as controls.