Distinct subcellular localization of TBLR1-RARα fusion protein. (A) Immunoblotting analysis of cytoplasmic and nuclear components of 293T cells transfected with Flag-tagged TBLR1-RARα. TBLR1-RARα was predominantly expressed in nucleus and a small amount of it in cytoplasm and was downregulated both in nucleus and cytoplasm after treatment with 1 μM ATRA. (B) Immunofluorescence analysis of 293T cells transfected with expression plasmids of vehicle (vector), PML-RARα-Flag, RARα-Flag, and TBLR1-RARα-Flag, respectively. Flag antibody was used as primary antibody, and DAPI for nuclear staining. At least 100 cells were counted for localization analysis. (C) The expression of TBLR1-RARα was decreased in 1 μM ATRA–treated 293T cells by immunoblotting. β-actin and H3 were used as internal controls for cytoplasmic and nuclear proteins. (D) Immunofluorescence analysis of 293T cells incubated with 1 μM ATRA for 48 hours. Bars (B,D) represent 8 μm. C, cytoplasm; N, nucleus.