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Fig. 1. Cellular distribution of endogenous and wt FLAG-TDP-43. (A) Diagram of wt TDP-43 showing the mutations introduced in the nuclear localization signal (NLS1 and NLS2). Other distinguishing features of TDP-43 are the two RRM domains (RRM-1 and RRM-2) and the putative nuclear export signal (NES, light green). (B) Distribution of wt TDP-43 and mutant TDP-43 in the nucleus (N) and cytoplasm (C), in non-transfected (endogenous) HeLa cells and HeLa cells and transiently transfected with FLAG-tagged wt and Myc-tagged mutant TDP-43. The detection of p84 and tubulin was carried out as control for nuclear and/or cytoplasmic contamination of the two fractions. (C) Indirect immunofluorescence of U2OS cells non-transfected (endogenous) and transiently transfected with FLAG-TDP-43. wtTDP-43 showed nuclear localization with foci formation, whereas – as expected – disruption of either NLS resulted in cytoplasmic accumulation of the mutants. Similar results were obtained in HeLa cells. The endogenous protein was visualized using anti-TDP-43 polyclonal antibody (ProteinTech) and FITC-conjugated secondary antibody; transfected TDP-43 was detected using either anti-FLAG or anti-Myc monoclonal antibodies and Texas-Red-conjugated secondary antibody. The nuclear membrane was seen using either anti-lamin A/C polyclonal antibody or anti-LAP2β polyclonal antibody, and Texas-Red- or FITC-conjugated secondary antibodies. Scale bars: 10 µm.
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