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. 2019 Apr 1;28(15):2459–2476. doi: 10.1093/hmg/ddz062

Figure 3.

Figure 3

Characterization of differentiated SH-SY5Y cells with inducible expression of TDP-43-Q331K. (A) pCW-TDP-43-Q331K vector construct containing Dox-inducible 1× FLAG-tagged WT or 1× FLAG-tagged Q331K mutation. (B) Immunoblots confirming comparable FLAG and TDP-43 expression after Dox induction. GAPDH served as loading control. (C) IF with FLAG antibody 96 h after induction shows substantially increased cytoplasmic localization of Q331K in comparison to WT (Scale bar, 10 μm). The 2.5-dimensional view of the localization is shown in the image below. (D) IF of staining with FLAG antibody shows increased cytosolic accumulation of mutant TDP-43 in cells expressing the Q331K compared to WT. Left panel shows DAPI staining. Second from the left panel shows FLAG staining. Third panel shows Phalloidin staining as a cytoplasmic marker. Right panel shows merged image (Scale bar, 10 μm). (E) Immunoblots confirming nuclear exclusion of mutant TDP-43 as compared to WT. Histone 3 served as a nuclear loading control. (F) PLA using TDP-43 and FLAG antibody and foci quantitation showed the accumulation of cytosolic foci in cells expressing Q331K compared to WT cells (Scale bar, 10 μm). **P<0.05.