FIGURE 3.

Expression of TDP-43-ΔNLS mutants led to sequestration of endogenous nuclear TDP-43. A, TDP-43 immunohistochemistry in a hippocampal section from a FTLD-U brain. Note clearing of nuclear TDP-43 (arrowheads) in neurons containing cytoplasmic TDP-43 inclusions, as compared with normal neurons (asterics). B, schematic diagram of N-terminal Myc-tagged TDP-43 protein highlighting the location of a bipartite NLS and NES. Both sets of basic aa (red) in NLS were mutated to generate three defective mutants (ΔNLS1, ΔNLS2, and ΔNLS1/2), and both sets of hydrophobic aa (blue) in NES were mutated to generate ΔNES1 and ΔNES2. C-K, double labeling of TDP-43 (red), Myc (green), and counterstaining with DAPI (blue) for nuclei. C-E, QBI-293 cells 72 h after transfection with Myc-TDP-43-WT (WT) and merged image (E) show colocalization of TDP-43 and Myc in nuclei of transfected cells. F-H, QBI-293 cells 24 h after transfection of Myc-TDP-43-ΔNLS1 (ΔNLS1). The merged image (H) shows colocalization of TDP-43 and Myc in the cytoplasm of transfected cells. I-K, QBI-293 cells 72 h after transfection of ΔNLS1. The merged image (K) shows colocalization of TDP-43 and Myc in the cytoplasm of transfected cells. Note the clearing of endogenous nuclear TDP-43 in cells expressing NLS1, as compared with nontransfected cells. Scale bars, 25 μm. ^*, nontransfected cells.