TDP-43 clearance and aggregation depend on ESCRT function. (A) Indicated strains were transformed with TDP-43-mRuby2 plasmid (pRB194) and grown to mid-logarithmic growth phase, and TDP-43-mRuby2 was detected by western blot using anti–TDP-43 antibody. TDP-43 levels were normalized to GAPDH loading control. **, P < 0.01; ***, P < 0.001 by one-way analysis of variance with Tukey’s post hoc test; N = 3. (B) Indicated strains were transformed with TDP-43-mRuby2 plasmid and imaged in mid-logarithmic growth phase with foci/cell quantification below. ***, P < 0.001 by one-way analysis of variance with Tukey’s post hoc test; N = 3. Scale bar, 5 µm. (C) Indicated strains were transformed with TDP-43-mRuby2 plasmid and grown to mid-logarithmic growth phase before being treated with 0.2 mg/ml cycloheximide for 0, 6, 12, and 24 h. TDP-43-mRuby2 levels were detected by western blot using anti–TDP-43 antibody and normalized to GAPDH loading control. **, P < 0.01; ***, P < 0.001 by two-way analysis of variance with Tukey’s post hoc test; N = 3. (D) Strains endogenously expressing GFP-Vps28, GFP-Vps36, and GFP-Vps4 were transformed with a TDP-43-mRuby2 plasmid and imaged at mid-log phase growth. Percentage values indicate colocalization frequency of TDP-43-mRuby2 foci with GFP foci. N = 3; scale bar, 5 µm. Error bars in all graphical panels represent standard error of the mean. Source data are available for this figure: SourceData F2.