FLNA interacts with TIF-90 and prevents TIF-90 enhancement of rRNA synthesis. (A) Interaction of FLNA and TIF-IA in primary AML cells (left). AML lysate (500 μg) combined from 10 samples was used for immunoprecipitation experiments. Cell lysate was immunoprecipitated with anti-IgG or anti-FLNA antibody and immunoblotted with anti-TIF-IA antibody (left). Interaction of TIF-FL and TIF-90 with FLNA (right). The 293T cells were cotransfected with green fluorescent protein (GFP)-FLNA or vector control, Myc-TIF-FL, or Myc-TIF-90. FLNA protein was immunoprecipitated with anti-GFP antibody and immunoblotted with anti-Myc antibody. (B) Mapping the interaction domain of FLNA for TIF-90. Schematic structure of FLNA full length and fragments (left); interaction of GST-tagged TIF-90 with FLNA fragments in cell lysate (right). (C) Effects of FLNA expression on TIF-FL and TIF-90 enhancement of rRNA synthesis. The 293T cells were cotransfected with Myc-tagged TIF-FL or TIF-90 and vector control or GFP-FLNA. Myc-TIF protein was immunoprecipitated with anti-Myc antibody and immunoblotted with anti-Pol I antibody (left); 5′ETS pre-rRNA and RNA labeling (middle); MTT and colony-forming assay (right). (D) Effects of FLNA full length and fragments on TIF-90 enhancement of rRNA synthesis. The 293T cells were cotransfected with Myc-TIF-90 and the indicated FLNA constructs. FLNA protein was immunoprecipitated with anti-GFP antibody and immunoblotted with anti-Myc antibody (left, top row); TIF-90 protein was immunoprecipitated with anti-Myc antibody and blotted for Pol I binding (left, second row); 5′ETS pre-rRNA and RNA labeling (middle); MTT and colony-forming assays (right).