American Society of Hematology

Figure 2.

$Figure 2. Flt3-ITD inhibits the interaction between SMRT and transcriptional repressors. (A) SMRT and PLZF mammalian 2-hybrid interaction is inhibited by Flt3-ITD overexpression. Fold activation was determined by comparing normalized luciferase activity obtained when the Gal4-TK-LUC reporter gene was transfected with Gal4-1-147 alone compared with the activity obtained when Gal4-PLZF and VP16-SMRT were coexpressed. The data are the mean (± standard deviation) of 8 independent experiments. (B) Coimmunoprecipitation interaction between PLZF and SMRT is inhibited by Flt3-ITD. Cells were transfected with full-length PLZF and SMRT expression plasmids in the presence (Flt3-ITD) or absence (vec) of Flt3-ITD expression plasmid. SMRT was precipitated by polyclonal goat anti-SMRT antibody (Santa Cruz Biotechnology; N-20) and the resulting fraction was immunoblotted by PLZF. Intensity of each band was quantified by NIH image 1.60 software. To measure the effect of Flt3-ITD, relative density was obtained from the density of the PLZF band in Flt3-ITD-transfected cells divided by that from vector-transfected cells. (C) Flt3-ITD overexpression inhibits the mammalian 2-hybrid interaction between SMRT and ETO. Experiment performed similar to panel A. Gal4-1-147 fused to full-length ETO plasmid was used as a 2-hybrid bait. The data are the mean (± standard deviation) of 8 independent experiments. (D) Flt3-ITD inhibits interaction between ETO and SMRT. Experiment performed similar to panel B. Full-length HA-tagged ETO and SMRT expression plasmids were transfected into 293T cells in the presence or absence of Flt3-ITD. SMRT was precipitated and the Western blot was performed by anti-HA antibody to detect ETO.$

Flt3-ITD inhibits the interaction between SMRT and transcriptional repressors. (A) SMRT and PLZF mammalian 2-hybrid interaction is inhibited by Flt3-ITD overexpression. Fold activation was determined by comparing normalized luciferase activity obtained when the Gal4-TK-LUC reporter gene was transfected with Gal4-1-147 alone compared with the activity obtained when Gal4-PLZF and VP16-SMRT were coexpressed. The data are the mean (± standard deviation) of 8 independent experiments. (B) Coimmunoprecipitation interaction between PLZF and SMRT is inhibited by Flt3-ITD. Cells were transfected with full-length PLZF and SMRT expression plasmids in the presence (Flt3-ITD) or absence (vec) of Flt3-ITD expression plasmid. SMRT was precipitated by polyclonal goat anti-SMRT antibody (Santa Cruz Biotechnology; N-20) and the resulting fraction was immunoblotted by PLZF. Intensity of each band was quantified by NIH image 1.60 software. To measure the effect of Flt3-ITD, relative density was obtained from the density of the PLZF band in Flt3-ITD-transfected cells divided by that from vector-transfected cells. (C) Flt3-ITD overexpression inhibits the mammalian 2-hybrid interaction between SMRT and ETO. Experiment performed similar to panel A. Gal4-1-147 fused to full-length ETO plasmid was used as a 2-hybrid bait. The data are the mean (± standard deviation) of 8 independent experiments. (D) Flt3-ITD inhibits interaction between ETO and SMRT. Experiment performed similar to panel B. Full-length HA-tagged ETO and SMRT expression plasmids were transfected into 293T cells in the presence or absence of Flt3-ITD. SMRT was precipitated and the Western blot was performed by anti-HA antibody to detect ETO.

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