NUP98-TOP1 fusion protein demonstrates nuclear localization. (A) Schematic representation of NUP98, TOP1, and NUP98-TOP1 proteins. Fusion breakpoints are indicated with vertical arrows. Functional domains of NUP98 include FXFG repeats (▨), GLEBS domain (□), ribonucleoprotein (RNP)-binding domain (▥), and nuclear localization signals (NLSs) (▦). TOP1 functional domains comprise N-terminal NLS, core domain (▪), linker (□), and C-terminus (). (B) Western blot analysis of total cell lysates from calcium-phosphate-transfected 293T cells detected by anti-GFP antibody. Cells were transfected with GFP-fusion constructs as described in “Materials and methods.” Arrows indicate the size of the expected full-length NUP98-TOP1 protein (150 kDA) and a smaller processed fragment. (C) Fluorescent microscopy images of 293T cells transfected as in panel B. Panels i, iv, vii, and x depict cells stained with 4′, 6-diamidino-2-phenylindole, dilactate (DAPI) for visualization of nuclei; panels ii, v, viii, and xi show visualization of GFP expression; panels iii, vi, ix, and xii show the overlay of DAPI and GFP. Top row (i-iii) results with empty pEGFP-C1 vector, demonstrating pan-cellular GFP expression in nucleus and cytosol. Second row (iv-vi) results with GFP-NUP98-TOP1 fusion, showing that NUP98-TOP1 directs nuclear expression. Third row (vii-ix) GFP-NT-Y723F also exhibits nuclear localization. Bottom row (x-xii) results with GFP-SH2-containing inositol-5-phosphatase (SHIP) used as positive control for cytosolic localization. (D) Retroviral vectors used to express NUP98-TOP1 and NT-Y723F in murine bone marrow. The expected sizes of full-length proviral transcripts are indicated. LTR indicates long terminal repeats; GFP, green fluorescent protein; IRES, internal ribosomal entry site.