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. 2020 Jan 27;130(3):1139–1155. doi: 10.1172/JCI130988

Figure 6. sTDP43 overexpression leads to the cytoplasmic deposition and nuclear clearance of endogenous TDP43.

Figure 6

(A) HaloTag fusions of flTDP43 or sTDP43 were expressed in HEK293T cells and immunoprecipitated with HaloLink. Bound TDP43 was immunoblotted (IB) with a C-terminal TDP43 antibody. GAPDH served as a loading control. (B) Quantification of data shown in A, demonstrating the fraction of total TDP43 bound to flTDP43-Halo, sTDP43-Halo, or Halo alone. Data were combined from 3 replicates. *P < 0.05, **P < 0.01 by 1-way ANOVA with Dunnett’s post hoc test. (C) HEK293T cells were transfected with EGFP or EGFP-tagged sTDP43, and then immunostained using an antibody that recognizes the endogenous TDP43 C-terminus (Endo). Red, nuclear regions of interest (ROIs) determined by DAPI staining. (D) Nuclear, endogenous TDP43 is reduced by sTDP43 overexpression in HEK293T cells. EGFP n = 1537, sTDP43-EGFP n = 1997, 3 replicates. ****P < 0.0001 by 2-tailed t test. (E) Cytoplasmic endogenous TDP43 is elevated by sTDP43 overexpression in HEK293T cells. EGFP n = 129, sTDP43-EGFP n = 113, 3 replicates. ****P < 0.0001 by 2-tailed t test. (F) Primary mixed rodent cortical neurons were transfected with EGFP or EGFP-tagged sTDP43, and then immunostained using a C-terminal TDP43 antibody. Red, nuclear ROIs determined by DAPI staining. (G) sTDP43 overexpression resulted in a significant drop in nuclear, endogenous TDP43 in primary neurons (EGFP n = 395, EGFP-sTDP43 n = 323, 3 replicates; ****P < 0.0001 by 2-tailed t test), but this was not accompanied by increases in cytoplasmic, endogenous TDP43 (H) (EGFP n = 394, EGFP-sTDP43 n = 323, 3 replicates, NS by 2-tailed t test). Scale bars: 20 μm (C and F).