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Fig. 2 | Acta Neuropathologica Communications

Fig. 2

From: Mice deficient in the C-terminal domain of TAR DNA-binding protein 43 develop age-dependent motor dysfunction associated with impaired Notch1−Akt signaling pathway

Fig. 2

TDP-∆C protein is predominantly localized in cytoplasm, and is less stable than TDP-43. a Schematic diagrams of wild-type TDP-43 (TDP-WT) and TDP-∆C proteins. The N-terminal domain of TDP-43 (a.a. 1–273), including the nuclear localization signal (NLS) and the nuclear export signal (NES), remains intact in TDP-∆C protein. Anti-TDP-43(N-terminal) antibody was raised against the extreme N-terminal domain (a.a. 1–10), whereas anti-TDP-43(A260) and anti-TDP-43(3H8) recognizes the region harboring a.a. 260 and the C-terminal domain (a.a. 274–414), respectively. Validation results for these antibodies are shown in Additional file 1: Figure S1. b Expression levels of TDP-WT and TDP-∆C proteins in spinal cord, cerebral cortex, cerebellum, and liver of wild-type (WT) and TDP-∆C (∆C) mice. Immunoblotting analyses using anti-TDP-43(3H8) antibody, that specifically recognizes TDP-WT, and anti-FLAG, specific to TDP-∆C, in the indicated tissues of 5-month-old WT and ∆C mice. c Representative immunofluorescent images of anterior horn (AH) in lumbar spinal cord (LSC) sections of 5-month-old WT and TDP-∆C (∆C) mice stained with anti-TDP-43(3H8) or anti-FLAG antibody. Scale bars: 20 μm. d Subcellular fractionation of LSC and cerebral cortex from 5-months-old TDP-∆C mice. Immunoblotting analyses of cytosolic and nuclear fractions from the indicated tissues of TDP-∆C mice using antibodies for TDP-43(3H8), FLAG, fibrillarin, and heat shock protein 110 (Hsp110). Note that endogenous TDP-WT and TDP-∆C protein were predominantly localized in nucleus and cytosol, respectively. e-g Immunoblotting analyses of endogenous TDP-43 and TDP-∆C proteins in brain and whole spinal cords (SC) detected by anti-TDP-43 antibody recognizing the amino acids near Ala 260 (A260) of TDP-43 or anti-FLAG antibody (e). A filled arrowhead indicates endogenous murine TDP-43, and open arrowheads indicate TDP-∆C, respectively. An asterisk denotes a non-specific band. Quantification of TDP-∆C relative to endogenous TDP-43 (f) or endogenous TDP-43 levels normalized to GAPDH (g) are plotted. Note that reduction of TDP-∆C protein levels was observed in both spinal cords and brains (f). While, the levels of endogenous TDP-43 protein were not affected by TDP-∆C (g). h and i Cycloheximide (CHX) chase assay revealed that TDP-∆C protein is less stable than TDP-WT protein. Mouse neuroblastoma Neuro2a (N2a) cells were transfected with expression plasmids for 3 × FLAG-tagged wild-type human TDP-43 (TDP-43(WT)-FLAG) or 3 × FLAG-tagged human TDP-43 mutant devoid of its C-terminal domain (TDP-∆C), and treated with CHX (15 μg/mL) for the indicated times. Cell lysates were then prepared and subjected to immunoblotting. Representative immunoblots using anti-FLAG and anti-β-actin antibodies are shown (h). Quantification of TDP-43(WT)-FLAG and TDP-∆C immunoblots relative to 0 h are plotted as mean ± standard error of the mean (SEM) (n = 3) Indicated p-values are results of multiple comparison t-test between TDP-43(WT) and TDP-∆C at the same time points (i)

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