Figure 1.

Molecular analysis of TDP-43 WT, S375G, and S375E stably expressing cells.A, Western blot analysis of the three TDP-43 stable clones’ expression upon tetracycline induction at three different time points: 24, 48, and 72 h. The WT, S375G, and S375E proteins are specifically detected with α-FLAG antibody. Tubulin is used as a standard loading control. B, stable clones endogenous TDP-43 autoregulation at 48 h. In the upper part of the panel, Western blot analysis was performed against total TDP-43 detecting both the stably expressed protein and the endogenous TDP-43. Tubulin was used as a loading control. Western blot quantification of endogenous TDP-43 expression is plotted in a column graph (red for WT, blue for S375G, and green for S375E samples). The statistical analysis of endogenous TDP-43 expression levels was performed with multiple comparison one-way ANOVA test with Bonferroni’s correction using GraphPad software (GraphPad Software, Inc) and is reported in the lower panel. C, endogenous POLDIP3 pre-mRNA splicing analysis in stably expressing cells was carried out with Qiaxcel capillary electrophoresis (upper panel). In this experiment, POLDIP3 exon 3 inclusion is plotted in a column graph (red for WT, blue for S375G, and green for S375E). Statistical analysis from three independent experiments was performed with multiple comparison one-way ANOVA test with Bonferroni’s correction using GraphPad software. D, solubility test on WT-, S375G-, and S375E-expressing clones. Western blot analysis was performed against total TDP-43 in endogenous conditions (left) as well as upon sodium arsenate treatment (right). E, conformational ¹³Calpha chemical shifts (top panel) for WT, S375G, and S375E TDP-43 CTD, calculated as ¹³Calpha (experimental) − ¹³Calpha (coil), where the latter are determined from the primary sequence using the parameters reported by Kjaergaard and Poulsen (103). Values higher than 0.3 ppm are indicative of partially populated alpha helix. Longitudinal relaxation rates (bottom panel) in the rotating frame for TDP-43 WT and S375 variants. CTD, C-terminal domain; TDP-43, TAR DNA-binding protein 43.