FIG 2.

Ezrin interacts with DDX3, and the antiezrin compound NSC305787 inhibits this interaction. (A) Interaction of ezrin with DDX3 in K7M2 mouse (left) and MG63.3 human (right) OS total cell lysates (TCL) by co-IP experiments. Protein complexes were immunoprecipitated from the cell lysates with antiezrin antibody (Ab) or negative-control total mouse IgG and immunoblotted (IB) with anti-DDX3 antibody. (B) K7M2 cells were treated with 3.0 μM NSC305787 for 8 h. Total cell lysates were immunoprecipitated with control IgG or an ezrin antibody, which was followed by Western blotting for ezrin and DDX3. (C) (Top) Recombinant wild-type ezrin and the phosphomimicking ezrin T567D mutant were expressed in bacteria and purified by cation-exchange chromatography on an SP Sepharose column, followed by adsorption chromatography on a heparin column. DDX3 protein was expressed in baculovirus-infected insect cells and purified by Ni²⁺ affinity column chromatography. Approximately 1.5 μg of each protein was run on a gel and stained with Coomassie blue. (Bottom) A purified DDX3 sample (1.5 μg) was run on a gel and stained with Coomassie blue (Coom.) (left), or 250 ng of purified DDX3 was run on a gel and transferred to a membrane for Western blot analysis using anti-DDX3 antibody (right). (D) Recombinant wild-type ezrin or the phosphomimicking ezrin mutant was immobilized on a CM5 chip in Biacore T-200. DDX3 was injected over the chip surface at six different concentrations (1.25, 2.5, 5.0, 10, 20, and 40 nM) in triplicate. The colored lines show real data, and the black lines represent curves fitted for a 1:1 binding model. (E) Equal amounts of lysates from K7M2 mouse OS cells were subjected to co-IP with antiezrin antibody. The resulting coprecipitates were treated with 0.5 mg/ml RNase A at 37°C for 30 min. −RNase A and no treat., samples that were incubated in the absence of RNase A for 30 min at 37°C or at 4°C, respectively. Immunoblotting was performed with anti-DDX3 and antiezrin antibodies.