Fig. 6: Aggresomal proteins are degraded via different pathways.

a HeLa cells treated with Btz (1 μM, 8 h) and recovered for 0 h, 15 h, and 21 h were lysed and subjected to a pulldown of ubiquitylated proteins using OtUBD beads. Input and pulldown samples were immunoblotted for ubiquitin, AMOTL2, and PLEK2; the Coomassie-stained membrane served as a loading control (CBB). Representative result from two independent experiments. b HeLa cells treated with Btz (1 μM, 8 h) and harvested after 15 h recovery, or recovered for six additional hours in the absence or presence of 1 μM Btz, 1 μM CB-5083, 10 μM VER, or 100 nM Baf were lysed in RIPA buffer. Untreated cells served as negative controls. RIPA-insoluble fractions were immunoblotted for PLEK2; the Coomassie-stained membrane served as a loading control (CBB). Shown is a representative western blot of four independent biological replicates. c Quantification of insoluble PLEK2 levels in b. Bars represent the mean fold change of insoluble PLEK2 relative to the levels at 15 h recovery ± SD (n = 4 biological replicates). One-way ANOVA. d HeLa cells treated with Btz (1 μM, 8 h) were recovered for 15 h and incubated with 100 nM Baf for six additional hours prior to cell lysis and isolation of an autophagosome-enriched fraction. The fractions of the isolation protocol depicted in Supplementary Fig. 7c were immunoblotted for PLEK2; the Coomassie-stained membrane served as a loading control (CBB). Representative result from two independent experiments. e Immunoblot analysis of autophagosome-enriched fraction (P1) after Proteinase K treatment with the indicated antibodies. Coomassie-stained membranes served as loading controls (CBB). Representative result from three independent experiments. f HeLa Flp-In TRex cells expressing GFP-TDP-43mut were treated with Btz (1 μM, 6 h) and fixed after 24 h recovery, or recovered for 14 additional hours in the absence or presence of 0.5 μM CB-5083 or 100 nM Baf, followed by immunostaining against ubiquitin and LC3. Scale bars, 50 μm. In the bottom row, arrowheads highlight GFP-, LC3-, and ubiquitin-positive structures. g Expression of endogenous TDP-43 and GFP-TDP-43mut in HeLa Flp-In TRex cells that were left untreated or were treated for 6 h with Btz was analyzed by immunoblot. Representative result from two independent experiments. h Quantification of the percentage of cells with a TDP-43-positive aggresome in f. Shown is the mean ± SD from n = 3 biological replicates with ≥150 cells per condition and replicate. One-way ANOVA. i Same as h, but quantification of total TDP-43-positive aggregate area.