GATA-2 interacts with HDAC3 in vitro.

(A) [³⁵S]-methionine-labeled HDAC1 or HDAC3 translated in vitro was incubated with GST–GATA-2 fusion protein or GST protein alone and pulled down with GST-agarose beads. After 4 washes, the precipitates were dissolved by SDS-PAGE and visualized by autoradiography. Input represents 4% of in vitro–translated HDAC proteins used for the pull-down assay. (B) [³⁵S]-labeled full-length HDAC3 was incubated with GST fusion proteins containing the various portions of GATA-2 as indicated in the upper panel. The precipitates were visualized by autoradiography following SDS-PAGE. Input represents 4% of in vitro–translated HDAC3 protein used for the pull-down assay. N-Zf indicates amino-terminal zinc finger; C-Zf, carboxyl-terminal zinc finger. (C) Various HDAC3 deletion constructs as indicated in the upper panel were produced by in vitro translation reaction and subjected to pull-down analysis by GST–GATA-2. Input represents 4% of each in vitro– translated HDAC3 protein used for the pull-down assay. (D) Various HDAC3 deletion constructs were transfected in COS cells. Whole cell lysates were extracted and subjected to pull-down by GST–GATA-2. After 4 washes the precipitates were subjected to immunoblot analysis with anti–X-press antibody. Inputs represent 5% of whole cell lysates used for each pull-down assay applied to confirm whether each HDAC3 was highly expressed.