Journal: Biomolecules
Article Title: Human Mutant Dynactin Subunit 1 Causes Profound Motor Neuron Disease Consistent with Possible Mechanisms Involving Axonopathy, Mitochondriopathy, Protein Nitration, and T-Cell-Mediated Cytolysis
doi: 10.3390/biom15121637
Figure Lengend Snippet: T cells invade the spinal cord parenchyma of mutant-DCTN1 tg mice. ( A ) Localization of Fas-positive cells (thin arrows) in ventral horn of wildtype DCTN1 transgenic mouse. Scale bar (same for ( B )) = 40 µm. ( B ) Fas-positive cells (thin arrows) in the ventral horn of mutant DCTN1 tg mice appear more numerous than in wildtype tg mice. Inset is dashed square showing Fas-positive cells in the vicinity of a motor neuron. Inset scale bar = 12 µm. ( C ) Fas-positive cells (thin arrows) near degenerating motor neurons (asterisk) in mutant-DCTN1 mice. Scale bar = 7 µm. ( D ) Box plot showing the mean Fas-positive cell number (with IQR and 5–95 percentile whiskers, n = 6/group) in the ventral horn of age-matched non-tg, human wildtype DCTN1 (p150) and human mutant DCTN1 (p150) at 8 months of age. ( E ) Infiltrated CD8-positive cells (arrows) in the ventral horn of mutant DCTN1 mice. CD8 immunoreactivity is also present in the surrounding white matter (wm). Scale bar = 12 µm. ( F ) Focal accumulations of CD8 immunoreactivity were seen in the ventral root exit zones in the ventral funiculus of mutant DCTN1 mice. Scale bar = 15 µm.
Article Snippet: The sections were blocked and permeabilized (1 h) in 10% normal goat serum (NGS) with 0.4% Triton-x 100 and then incubated (4 °C) in primary IgG antibodies overnight: rabbit monoclonal anti-cleaved caspase-8 (1:500, D5B2), rabbit monoclonal anti-cleaved caspase-3 (1:4000, D3E9), rabbit polyclonal anti-SOD2 (1:2000), rabbit polyclonal anti-Parkin (1:500), rabbit polyclonal anti-mouse TNF-α (Chemicon, St. Louis, MO, USA, 1:500), rabbit monoclonal anti-IL9 (Abcam, Cambridge, MA, USA, 1:500), monoclonal anti-nitrated Hsp90 (1:1000), a mouse monoclonal anti-Fas (BD Transduction Laboratories, Franklin Lakes, NJ, USA, 1:500), and a rabbit polyclonal antibody to CD8 (Proteintech, 1:500).
Techniques: Mutagenesis, Transgenic Assay
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