Figure 2. Detection of stress-induced... | Rockefeller University Press

Figure 2.

$Figure 2. Detection of stress-induced TDP-43 oligomerization using the luciferase split assay. (A) Immunofluorescence of endogenous TDP-43 after 400-mM sorbitol treatment in HEK-293 cells at various time points. Stress granules were stained by coimmunolabeling with a TIAR antibody, and nuclei were stained using DAPI. After 1 h of sorbitol treatment, nucleocytoplasmic redistribution of TDP-43 and accumulation in stress granules is observed. Bar, 10 µm. (B) Sorbitol treatment induces the formation of insoluble TDP-43 as assessed by immunoblotting of soluble and insoluble cell lysate fractions. (C) Luciferase activity measurement of nuclear/cytoplasmic fractions of TDP-Luc–transfected HEK-293 cells after 1 h of sorbitol-induced stress. n = 11 per group. (D) SEC of cell lysates derived from TDP-L1 + TDP-L2–transfected HEK-293 cells after sorbitol-induced stress. Absorbance at 280 nm shows the size distribution of the total protein content. (E) Luciferase activity measurement (top) and TDP-43–specific dot blot analysis (bottom) in the SEC fractions of stressed HEK-293 cells in D. (F) Total luciferase activity measurement in living HEK-293 cells expressing TDP-L1 and TDP-L2 with or without sorbitol stress. n = 20 per group. Mean ± SEM. **, P < 0.01; ***, P < 0.001. The data shown in D and E represent single SEC runs representative for at least three independent repeats. mAU, milli absorbance units.$

Detection of stress-induced TDP-43 oligomerization using the luciferase split assay. (A) Immunofluorescence of endogenous TDP-43 after 400-mM sorbitol treatment in HEK-293 cells at various time points. Stress granules were stained by coimmunolabeling with a TIAR antibody, and nuclei were stained using DAPI. After 1 h of sorbitol treatment, nucleocytoplasmic redistribution of TDP-43 and accumulation in stress granules is observed. Bar, 10 µm. (B) Sorbitol treatment induces the formation of insoluble TDP-43 as assessed by immunoblotting of soluble and insoluble cell lysate fractions. (C) Luciferase activity measurement of nuclear/cytoplasmic fractions of TDP-Luc–transfected HEK-293 cells after 1 h of sorbitol-induced stress. n = 11 per group. (D) SEC of cell lysates derived from TDP-L1 + TDP-L2–transfected HEK-293 cells after sorbitol-induced stress. Absorbance at 280 nm shows the size distribution of the total protein content. (E) Luciferase activity measurement (top) and TDP-43–specific dot blot analysis (bottom) in the SEC fractions of stressed HEK-293 cells in D. (F) Total luciferase activity measurement in living HEK-293 cells expressing TDP-L1 and TDP-L2 with or without sorbitol stress. n = 20 per group. Mean ± SEM. **, P < 0.01; ***, P < 0.001. The data shown in D and E represent single SEC runs representative for at least three independent repeats. mAU, milli absorbance units.

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