ER stress and proteasome inhibition cause redistribution of wildtype and ALS-linked mutant TDP-43.

<p>(<b>A</b>) Neuro2a cells were transfected with the construct indicated to the left of each series of panels (mCherry alone, wildtype TDP-43-mCherry, or mutant D169G, G294A, A315T, Q331K, M337V or N390D TDP-43-mCherry), and fixed at 48 h post-transfection. mCherry fluorescence is shown in the left panels, and Hoechst nuclei stain is shown in the merged right panels. Note the largely nuclear distribution of wildtype and mutant TDP-43-mCherry, but with low levels of non-nuclear mCherry fluorescence in some cells, indicated by arrows. (<b>B</b>) Immunoblot of cell lysates expressing mCherry alone or wildtype or mutant TDP-43-mCherry showing equal expression levels of all proteins, and levels of endogenous (End.) TDP-43. Approximate molecular weight markers are shown on the right. (<b>C</b>) Quantification of the effect of thapsigargin or MG132 treatment on the percentage of Neuro2a cells with cytoplasmic mCherry fluorescence. Results are expressed as mean ± SEM, <i>n</i> = 3, *p<0.05 versus respective vehicle treated controls by two-way ANOVA with Bonferroni's post-test. (<b>D</b>) Neuro2a cells were transfected as in <b>A</b> and treated with 100 nM thapsigargin for 24 h prior to fixation. (<b>E</b>) Neuro2a cells were transfected as in <b>A</b> and treated with 10 µM MG132 for 24 h prior to fixation. Note the increased non-nuclear distribution of wildtype and mutant TDP-43-mCherry with both thapsigargin and MG132 treatment, indicated by arrows. All scale bars represent 10 µm.