Fig. 3.
Usp8 interaction and colocalization with α-synuclein. (A) Wild-type (Usp8WT) or catalytically inactive (Cys786 to Ala) Usp8 was immunoprecipitated with untagged α-synuclein when expressed in HEK-293T cells. (B) Usp8 activity as measured by binding to HA-Ub-Br2 is not affected by increasing α-synuclein levels or expression of α-synuclein mutants. EV, empty vector; IP, immunoprecipitation; WCL, whole-cell extract. (C) Bimolecular fluorescence complementation signifying Usp8–α-synuclein interaction and expression of endosomal Rabs (Rab5, Rab7, and Rab11) tagged to a red fluorescent protein. Schematic depicting the bimolecular fluorescence complementation assay used in this study. Human Usp8 is tagged to the N-terminal fragment of Venus fluorescent protein (VFP) and α-synuclein is tagged to the C-terminal fragment of VFP. Quantification of the percentage overlap (Manders’ colocalization coefficient) between Usp8 and α-synuclein (green) with the indicated endosomal markers (red); n = 100 cells per condition; ***P < 0.001, one-way ANOVA. (D) Localization of Usp8 in relation to endosomal markers in human iPSc-derived dopaminergic neurons: triple labeling of neurons with TH (for detection of dopaminergic neurons; first column, blue), Usp8 (second column, green), and one of the following: (i) early endosome, EEA1; (ii) late endosome, LBPA; or (iii) α-synuclein (third column, red). Quantification of the percentage overlap (MCC) of Usp8 with the indicated markers in B; n = 50 neurons per condition. (Scale bar, 10 μm.) Error bars correspond to standard error of the mean.