Fig 1: Lysosomal damage in TDP-43 aggregates producing cells. Subcellular localization of Galectin 3 (Gal3) was evaluated by immunofluorescence. (A) Control HEK293 cell line without 1 mM of LLOMe for 2 h. (B) Control HEK293 cell line with 1 mM of LLOMe for 2 h. Green Gal3 puncta observed (left panel) were considered as lysosomal damage. Lamp2A positive lysosomes co-localization with Gal3 puncta were evaluated (Lamp2A; red and Gal3; green). Numbered arrows indicate Lamp2A positive lysosomes colocalizing with Gal3 puncta (B, right panel, yellow). (C) Subcellular localization of Gal3 was evaluated in the HEK293 cell line over-expressing Flag-TDP-12xQ/N for 72 h. Like in (B), the observed green Gal3 puncta were considered as lysosomal damage (left panels). Numbered arrows indicate Gal3 puncta surrounding Lamp2A positive lysosomes (C, right panel, yellow), whereas numbered arrows heads indicate Gal3 puncta colocalizing with Lamp2A lysosomes (C, right panels, yellow). Images are representative of at least three independent experiments. (D) Intensity line profiles of foci 1, 2, 4 and 5 showed in (C) were obtained using the plot profile plugin of ImageJ software. Data were normalized against maximum intensity values in each channel.
Fig 2: Aggregated-prone form of TDP-43 co-precipitates with Hsc70. (A) The HEK293 Flp-in, HEK293 Flag-TDP-43 WT and HEK293 Flag-TDP-12xQ/N cell lines were incubated with tetracycline for 72 h. Then, immunoprecipitation was performed using an Anti-Flag M2 Affinity Gel. Immunoprecipitated Flag-tagged proteins were analyzed by Western blot using an anti-Flag M2 antibody. (B) The presence of co-precipitated Hsc70 protein in samples shown in (A) was evaluated by Western blot using an anti-Hsc70 antibody. IN, inputs (20 µg of cell lysate); IP, immunoprecipitates. Western blots are representative from at least three independent experiments.
Fig 3: Wild type and aggregated-prone TDP-43 forms are present in lysosomal fractions isolated from cell culture. (A) The lysosomal fraction was isolated from cell line over-expressing Flag-TDP-43 WT during 72 h, then endogenous, as well as Flag-tagged protein was evaluated by Western blot. (B) The lysosomal fraction was isolated from cell line over-expressing Flag-TDP-12xQ/N during 72 h, then endogenous, as well as Flag-tagged protein was evaluated by Western blot. PSN, post-nuclear fraction; F1, post-nuclear fraction after 1st ultracentrifugation step; Mit/Lys, fraction containing mitochondria and lysosomes after 1st ultracentrifugation step; Mit, mitochondria fraction after 2nd centrifugation step; Lys, lysosomal fraction after 2nd centrifugation step. Western blots are representative from at least three independent experiments.
Fig 4: TAR DNA binding protein 43 kDa (TDP-43) is substrate of chaperone mediated autophagy (CMA) in vitro. (A) Schematic representation showing the isolation of lysosomal fractions from rat liver tissue and the subsequent analysis of samples by Western blot. (B) Lysosomal CMA+ and CMA- fractions were pooled, preincubated with or without proteases inhibitors (PI) and then recombinant TDP-43 was added at different concentrations (0, 0.5, 0.75, 1 and 2 µg). The presence of lysosomal associated membrane protein isoform 2 isoform A (Lamp2A) and TDP-43 was analyzed by Western blot. (C) Relative amounts of recombinant TDP-43 were quantified from at least three independent experiments and the input was used to normalize the samples. The proportion of TDP-43 in PI- and PI+ was calculated using normalized data. Numerical results are reported as mean ± SE. Differences among means were analyzed from three independent experiments using two-way ANOVA, followed by the Bonferroni post hoc test to determine statistical significance (p < 0.05). (D) Recombinant TDP-43 (1 µg) was preincubated with different amounts of recombinant glyceraldehyde-3-phosphate dehydrogenase protein (GAPDH; 0, 5, 10 and 15 µg) and subjected to lysosomal degradation. TDP-43, Lamp2A, and GAPDH were analyzed by Western blot. (E) Densitometric quantification of TDP43 protein levels showed in (D) were normalized by the Lamp2A signal. The up-take was considered as the difference between TDP-43 in PI+ and PI- (Up-take = PI+–PI-). Three independent experiments were quantified and analyzed by ordinary one-way ANOVA (p < 0.05). ns: no significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 5: TDP-43 as a substrate of CMA in vivo. (A) Lysosomal fractions CMA+ and CMA- were isolated from rat brains in a similar way as indicated in Figure 1A. The different fractions obtained were compared with fractions isolated from rat liver. (B) CMA+ and CMA- lysosomal fractions were analyzed by Western blot in order to evaluate the protein levels of endogenous TDP-43, Lamp2A, Heat shock cognate protein 70 kDa (Hsc70) and GAPDH. (C) Densitometric quantification of each protein was performed in CMA+ and CMA- lysosomal fractions. Densitometric data obtained from CMA+ and CMA- conditions were summed and taken as 100%. Using this data, the proportions of each protein in CMA+ and CMA- lysosomal were calculated. The calculated proportions from three independent experiments were used to obtain the final graph. Numerical results are reported as mean ± SE. Differences among means were analyzed from three independent experiments using two-way ANOVA, followed by the Bonferroni post hoc test to determine statistical significance (p < 0.05). ***p < 0.001, ****p < 0.0001.
Supplier Page from Abcam for Recombinant Human TDP43 protein