The SMRT-RID is critical to the ability of Flt3-ITD to inhibit SMRT-PLZF interaction. (A) The 293T cells were transfected with full-length PLZF and SMRT (1-1495) or the RID deletion mutant SMRT (1-926) expression plasmids in the presence (F) or absence (V) of Flt3-ITD expression plasmid. SMRT was precipitated by polyclonal goat anti-SMRT antibody and the immunoprecipitates were separated by electrophoresis and immunoblotted for PLZF. The intensity of each band was measured by densitometry using NIH image 1.60 software. Relative density of PLZF was determined as described in Figure 2. (B) Flt3-ITD does not increase phosphorylation of SMRT. The 293T cells were transfected with full-length GFP-SMRT (1-1495) with indicated expression plasmids: either control vector (vec), Flt3-ITD, or Ha-Ras plasmid. Cell extracts were then harvested in the presence or absence of calf intestinal alkaline phosphatase (CIP) and blotted with SMRTe polyclonal antibody.