NEDD4 regulates TDP-43 turnover, toxicity, and ubiquitination. (A) HEK-TDP-43-GFP cells were transfected with siRNAs targeting NEDD4 or noncoding control and grown in normal conditions for 3 days. Cells were then treated with 0.3 mg/ml cycloheximide for the indicated amounts of time, and TDP-43-GFP levels were detected by western blot with anti-GFP antibodies and normalized to the level of the GAPDH loading control. ***, P < 0.001 by two-way analysis of variance with Tukey’s post hoc test; N = 3. (B) HEK-TDP-43-GFP cells were transfected with NEDD4-mCherry plasmid (pRB334) and grown in normal conditions for 3 days. TDP-43-GFP levels were detected by western blot with anti-GFP antibodies and normalized to the level of the GAPDH loading control. *, P < 0.05 by Welch’s two-tailed T test; N = 3. (C) HEK293A, HEK-TDP-43-GFP, and HEK-TDP-35-GFP cells were seeded at equal cell numbers and transfected with NEDD4-mCherry, a mock transfection control (left), or siRNA targeting NEDD4 or noncoding control (right), and cell proliferation was compared after 48 h *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way analysis of variance with Tukey’s post hoc test; N = 3. (D) HEK-TDP-43-GFP cells were transfected with NEDD4-mCherry plasmid and incubated for 24 h before being imaged. Percentage value indicates colocalization frequency of cytoplasmic TDP-43-GFP foci with NEDD4-mCherry foci. N = 3; scale bar, 10 µm. (E) HEK-TDP-43-GFP cells were transfected with NEDD4-mCherry plasmid or a mock transfection and incubated for 48 h before mCherry was immunoprecipitated, and TDP-43-GFP binding was assessed via western blot using anti–TDP-43 antibodies. (F) HEK-TDP-43-GFP cells were transfected with siRNAs targeting NEDD4, NEDD4-mCherry plasmid, or a mock control and grown for 3 days before TDP-43-GFP was immunoprecipitated under denaturing conditions (See Materials and methods). Error bars in all graphical panels represent standard error of the mean. Source data are available for this figure: SourceData F5.