FIGURE 6.

ALS-associated TARDBP mutations do not alter the cellular localization of TDP-43. A, Myc-tagged wild-type TDP-WT or TDP-43 carrying the indicated ALS-associated point mutations (A315T, M337V, or G348C) were transiently transfected into HeLa cells. 24 h post-transfection cells were subjected to heat shock (44 °C for 1 h) or kept at control temperature (37 °C). Afterward cells were fixed, stained with a myc (green) and TIA-1 (red)-specific antibody and a nuclei counterstain (blue), and analyzed by confocal microscopy. Both TDP-WT and the ALS-associated point mutants were nuclear under control conditions and remained nuclear upon heat shock. Scale bars = 20 μm. B, shown are expression levels of TDP-43 constructs used in A. Total cell lysates were analyzed by immunoblotting with a myc-specific antibody (upper panel). Tubulin served as a loading control (lower panel). All lanes were from the same exposure of the same blot. C, quantification of nuclear and cytosolic fluorescence intensities of myc staining at 37 °C is shown. Error bars indicate S.D.