Figure 5.

Phosphorylated TDP-43 C-terminal fragment is resistant to proteasomal degradation. To calculate the half-life of GFP-TDP_220-414, cells were maintained in doxycycline-free media for 5 days. Doxycycline (1 μg/ml) was then added to the media to arrest GFP-TDP_220-414 expression. Cells were harvested immediately (0 hours) as well as 6, 12, 18 and 24 hours after the addition of doxycycline. (A) Western blot analysis of cell lysates using antibodies towards the C-terminus of TDP-43 (cTDP-43) or phosphorylated TDP-43 (pTDP-43). The arrow indicates GFP-TDP_220-414, the asterisk (*) endogenous TDP-43, and the dashed arrows cleavage products, likely generated from the truncation of GFP-TDP_220-414._. Dox=doxycycline, NS=non-specific. (B) Densitometric quantification of GFP-TDP_220-414 in cells was calculated by dividing the density of bands for total or phosphorylated GFP-TDP_220-414 by that of the corresponding GAPDH band, then normalizing each time-point to GFP-TDP_220-414/GAPDH levels at 0 hours. The half life (T1/2) of total GFP-TDP_220-414 was 14.2 hours; T1/2 for phospho-GFP-TDP_220-414 was 22.1 hours. ** represents P<0.001, as assessed by 1-way ANOVA, followed by Tukey's posthoc analysis (n = 3). (C) To examine the solubility of total and phosphorylated-GFP-TDP_220-414, M17D3 cells were incubated in doxycycline-free media for 6 days. Cell lysates were separated into Triton X-100-soluble (TS) and -insoluble (TIS) fractions and analyzed by Western blotting using anti-GFP and anti-pTDP-43 antibodies. (D) Densitometric quantification of total and phosphorylated GFP-TDP_220-414 in the Triton X-100-soluble and -insoluble fractions. Data was collected from 3 separate experiments and is shown as the mean+SEM.