Figure 1. Quantitative detection of... | Rockefeller University Press

$Figure 1. Quantitative detection of TDP-43 oligomers in living cells using a protein complementation approach. (A) TDP-43 split luciferase assay principle: oligomerization of TDP-43 monomers fused to nonbioluminescent G. princeps luciferase halves (L1 and L2) restores luciferase complementation, which can be quantified. (B) Western blot analysis of TDP-L1 and -L2 coexpression or TDP-Luc expression in HEK-293 cell lysates 48 h after transfection. (C) Luciferase activity measurement in living HEK-293 cells 72 h after cotransfection of either L1 + L2, L1 + TDP-L2, or TDP-L1 + TDP-L2. n = 10 per group. Mean ± SEM. ****, P < 0.0001. (D) SEC of cell lysates derived from TDP-L1 + TDP-L2–transfected HEK-293 cells. The molecular size standard proteins used were blue dextran (2,000 kD), thyroglobulin (669 kD), β-amylase (200 kD), and BSA (66 kD). (E) Luciferase activity measurement (top) and TDP-43–specific dot blot analysis (bottom) of the same SEC fractions shown in D. The data shown in D and E represent single SEC runs representative for at least three independent repeats.$

Figure 1.

Quantitative detection of TDP-43 oligomers in living cells using a protein complementation approach. (A) TDP-43 split luciferase assay principle: oligomerization of TDP-43 monomers fused to nonbioluminescent G. princeps luciferase halves (L1 and L2) restores luciferase complementation, which can be quantified. (B) Western blot analysis of TDP-L1 and -L2 coexpression or TDP-Luc expression in HEK-293 cell lysates 48 h after transfection. (C) Luciferase activity measurement in living HEK-293 cells 72 h after cotransfection of either L1 + L2, L1 + TDP-L2, or TDP-L1 + TDP-L2. n = 10 per group. Mean ± SEM. ****, P < 0.0001. (D) SEC of cell lysates derived from TDP-L1 + TDP-L2–transfected HEK-293 cells. The molecular size standard proteins used were blue dextran (2,000 kD), thyroglobulin (669 kD), β-amylase (200 kD), and BSA (66 kD). (E) Luciferase activity measurement (top) and TDP-43–specific dot blot analysis (bottom) of the same SEC fractions shown in D. The data shown in D and E represent single SEC runs representative for at least three independent repeats.

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