Fig. 3.

Tnks-1/2 regulates the degradation of TDP-43. (A) Cells were treated with the protein-synthesis inhibitor cycloheximide (CHX) in the presence of vehicle (DMSO) or the Tnks-1/2 inhibitor XAV939 (10 µM). The levels of endogenous TDP-43 were assessed by immunoblot. (B) Endogenous TDP-43 was less stable in the presence of XAV939 compared with DMSO-treated control. Mean±s.d. of three independent experiments. Two-way ANOVA (P<0.01) and Sidak's multiple comparison test. (C). Cells expressing TDP-43-WT-YFP and TDP-43-ΔTBD-YFP were treated with CHX, and protein levels were determined by immunoblot for TDP-43. (D) TDP-43-ΔTBD-YFP was less stable than TDP-43-WT-YFP upon CHX treatment. Mean±s.d. of three independent experiments. Two-way ANOVA (P<0.0001) and Sidak's multiple comparison test. (E) Cells expressing TDP-43-YFP (-WT, -ΔTBD, -R165A, -H166A, -M167A, -I168A, -D169A and -G1870A were treated with CHX and protein levels determined by immunoblot. (F) Mutations in the TBD of TDP-43-YFP that abolish the interaction with Tnks-1/2 (ΔTBD, H166A or I168A) led to a reduction in protein stability compared with TDP-43-WT. By contrast, the stability of TDP-43 with mutations in the TBD that do not affect the interaction with Tnks-1/2 (R165A, M167A, D169A or G170A) was no different from the stability of TDP-43-WT. NS, not significant; **P<0.01, ****P<0.0001.