Fig. 5. NDD-TDP-43 mutations induce phosphorylation, insolubilization, and coaggregation with WT TDP-43.

(A) Representative images of Neuro2a cells transiently expressing TDP-43 WT, NDD mutants, or NLS mutant immunostained with anti-FLAG and anti–pTDP-43. (B) Quantification of the percentage of the pTDP-43–positive cells per FLAG-positive cells in (A). Over 100 cells were quantified per experiment. (C) Representative immunoblots of Neuro2a cells transiently transfected with TDP-43-3×FLAG WT and mutants. (D) Quantification of pTDP-43/FLAG ratios (relative to TDP-43^WT) in (C). (E) Representative immunoblots of Neuro2a cells transiently expressing TDP-43-3×FLAG WT and mutants. Proteins sequentially extracted with tris-HCl (TS), 1% Triton X-100 (TX), and 1% sarkosyl (Sar), as well as a sarkosyl-insoluble fraction (ppt), were analyzed by immunoblotting. (F) Quantification of the percentage of each fraction relative to the total amount of TDP-43-3×FLAG in (E). (G) Representative immunoblots of the sequentially extracted lysates from Neuro2a cells transiently expressing TDP-43^6M-3×FLAG full-length (FL) and deletion mutants. (H) Quantification of the percentage of each fraction to the total amount of TDP-43-3×FLAG in (G). (I) Representative immunofluorescence images of Neuro2a cells transiently expressing TDP-43^6M-3×FLAG (top), TDP-43^WT-HA (middle), or both (bottom). Insets show magnified images of the outlined square area. (J) Line scan analysis of coaggregation of TDP-43^6M-3×FLAG and TDP-43^WT-HA in the inset from (I). (K) Representative immunoblots of the sequentially extracted lysates from Neuro2a cells transiently coexpressing TDP-43^WT-3×FLAG or TDP-43^6M-3×FLAG and TDP-43^WT-HA. (L) Quantification of the percentage of TDP-43-3×FLAG in each fraction relative to the total amount of TDP-43^WT-HA in (K). Scale bars, 10 μm (A and I). n = 3, biologically independent experiments (B, D, F, H, and L). Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 [ANOVA with Tukey’s test in (B), (D), (F), and (H) and unpaired two-sided t test in (L)].