Interaction of GATA-2 with PLZF in mammalian cells.

(A) Nuclear extracts of 293T cells transfected with expression plasmids encoding PLZF or Flag-GATA-2 were immunoprecipitated (IP) with anti-Flag antibody and were analyzed by Western blotting with anti-PLZF (top panel) or anti-Flag (lower panel) antibodies. Nonimmunoprecipitated material (10% input) was analyzed as a control for appropriate expression of proteins programmed by transfected plasmids. (B) Nuclear extracts of hematopoietic KG1 cells were immunoprecipitated with anti-PLZF antibody and analyzed by Western blotting with anti-PLZF (top panel) or anti-GATA-2 (lower panel) antibody. Input (10%) was used as a control for appropriate expression of the proteins. Numbers on the left indicate positions of molecular weight markers in kilodaltons. (C) Nuclear extracts of KG1 cells were incubated with biotinylated oligonucleotides harboring GATA motifs (wt GATA-oligo) or biotinylated mutant oligonucleotides in which GATA motifs were changed to TTTA (mut GATA-oligo). Oligonucleotides were then recovered by streptavidin–agarose beads, and the precipitated proteins were analyzed by Western blotting with anti-PLZF (top panel) or anti-GATA-2 (lower panel) antibody. Input (10% input) was used as a control. Note that GATA-2 and PLZF coprecipitated in transfected cells (A) and native hematopoietic cells (B). Endogenous GATA-2 had a capacity to recruit PLZF to GATA motifs in DNA (C).