Fig 2: USP8 treatment reverses LPS-induced USP8 degradation in the spleen. **P<0.01; ##P<0.01 (n=9/group). LPS, lipopolysaccharide; NC, negative control; USP8, ubiquitin-specific protease 8.

Fig 3: Enzymes of the ubiquitin system affect A53T α-synuclein aggregation.(A) Quantification of western blots (shown in S1 Raw images) showed reduction of USP13, USP8 and USP9X upon addition of specific pools of four siRNAs. The non-targeting (NT) negative control siRNAs were also pools of four siRNAs. (B) Quantification of total protein content in western blot samples showed no significant differences between treatments. (C) All samples were seeded with 0.1 μg/mL fibrils91. FRET assays for pS129-pS129 α-synuclein aggregation showed increased aggregation in response to USP8 and USP13 siRNAs and decreased aggregation in response to USP9X siRNAs. SCNA siRNAs was included as positive control and non-targeting (NT) siRNAs as negative control. (D) Electrochemiluminescence immunoassay showed down-regulation of α-synuclein protein levels in doxycycline treated neurons in response to SCNA and USP9X siRNAs. Data are shown as mean±SEM. Data in (A) and (B) were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test (n = 2, m = 2). Data in (C) and (D) were analyzed by one-way ANOVA followed by Sidaks’s multiple comparison test (n = 3, m = 6). ns = non-significant, *p<0.05 and **p<0.01 and ****p<0.0001.

Fig 4: USP8 treatment reverses structural damage to the spleen in LPS-induced mice, as visualized using hematoxylin and eosin staining. Representative images from different groups of mice are shown. Scale bar, 100 µm. LPS, lipopolysaccharide; NC, negative control; PALS, periarteriolar lymphoid sheath; USP8, ubiquitin-specific protease 8.

Fig 5: Alteration to mouse spleen morphology in the different groups. **P<0.01; #P<0.05 (n=5/group). LPS, lipopolysaccharide; NC, negative control; USP8, ubiquitin-specific protease 8.