Figure 2. Insoluble protein aggregates... | Rockefeller University Press

Figure 2.

$Insoluble protein aggregates accumulate in the absence of SETX. (A) Detergent-resistant aggregates were isolated in U2OS cells with SETX shRNA-mediated depletion or mock treatment as previously described (Lee et al., 2021). Whole lysates and detergent-resistant aggregates were isolated and analyzed by Western blotting for PSMD2, TDP-43, and PSMD8. (B) Quantification of PSMD2, full-length TDP-43 (FL), truncated TDP-43 (TDP35), and PSMD8 abundance in aggregate fractions normalized by lysate levels from three independent experiments, normalized to levels in control cells. (C) SH-SY5Y cells with control or SETX shRNA were induced or not induced to differentiate as indicated. Western blotting was performed to measure the levels of PSMD2, TDP-43, and PSMD8 in lysates and aggregates. (D) Levels of PSMD2, full-length TDP-43 (FL), truncated TDP-43 (TDP35), and PSMD8 in aggregate fractions normalized by lysate levels were quantified and shown relative to control cells. (E) U2OS cells with control or SETX shRNA were treated with PARP inhibitor veliparib (10 μM) as indicated. Levels of PSMD2, TDP-43, and PSMD8 were monitored by Western blotting in lysates and aggregate fractions. (F) Levels of PSMD2, full-length TDP-43 (FL), truncated TDP-43 (TDP35), and PSMD8 in aggregate fractions normalized by lysate levels were quantified in three independent experiments; shown relative to control cells. (G) Detergent-resistant aggregates were isolated in U2OS cells with SETX shRNA-mediated depletion with N-Acetyl cysteine (NAC, 1 mM) as indicated. Whole lysates and detergent-resistant aggregates were isolated and analyzed by Western blotting for PSMD2 and PSMD8. (H) Quantification of PSMD2 and PSMD8 abundance in aggregate fractions normalized by lysate levels from three independent experiments, normalized to levels in control cells. All P values are derived using a two-tailed t test assuming unequal variance, using three biological replicates (n = 3). Error bars indicate standard error. *, **, ***, and **** indicate P < 0.05, 0.01, 0.001, and 0.0001, respectively; ns = not significant. Source data are available for this figure: SourceData F2.$

Insoluble protein aggregates accumulate in the absence of SETX. (A) Detergent-resistant aggregates were isolated in U2OS cells with SETX shRNA-mediated depletion or mock treatment as previously described (Lee et al., 2021). Whole lysates and detergent-resistant aggregates were isolated and analyzed by Western blotting for PSMD2, TDP-43, and PSMD8. (B) Quantification of PSMD2, full-length TDP-43 (FL), truncated TDP-43 (TDP35), and PSMD8 abundance in aggregate fractions normalized by lysate levels from three independent experiments, normalized to levels in control cells. (C) SH-SY5Y cells with control or SETX shRNA were induced or not induced to differentiate as indicated. Western blotting was performed to measure the levels of PSMD2, TDP-43, and PSMD8 in lysates and aggregates. (D) Levels of PSMD2, full-length TDP-43 (FL), truncated TDP-43 (TDP35), and PSMD8 in aggregate fractions normalized by lysate levels were quantified and shown relative to control cells. (E) U2OS cells with control or SETX shRNA were treated with PARP inhibitor veliparib (10 μM) as indicated. Levels of PSMD2, TDP-43, and PSMD8 were monitored by Western blotting in lysates and aggregate fractions. (F) Levels of PSMD2, full-length TDP-43 (FL), truncated TDP-43 (TDP35), and PSMD8 in aggregate fractions normalized by lysate levels were quantified in three independent experiments; shown relative to control cells. (G) Detergent-resistant aggregates were isolated in U2OS cells with SETX shRNA-mediated depletion with N-Acetyl cysteine (NAC, 1 mM) as indicated. Whole lysates and detergent-resistant aggregates were isolated and analyzed by Western blotting for PSMD2 and PSMD8. (H) Quantification of PSMD2 and PSMD8 abundance in aggregate fractions normalized by lysate levels from three independent experiments, normalized to levels in control cells. All P values are derived using a two-tailed t test assuming unequal variance, using three biological replicates (n = 3). Error bars indicate standard error. *, **, ***, and **** indicate P < 0.05, 0.01, 0.001, and 0.0001, respectively; ns = not significant. Source data are available for this figure: SourceData F2.

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