Fig. 4.

α-Synuclein disease mutants are more susceptible to SUMOylation and aggregation. (A) In vitro SUMOylation was carried out by incubating His–α-synuclein WT and disease variants with SUMOylation components, with and without GST-PIAS2. Levels of His–α-synuclein SUMOylation were determined with anti–α-synuclein antibody (first and second panels). Third panel shows a short exposure to reveal α-synuclein levels. Graph depicts the percent of in vitro SUMOylated α-synuclein normalized to non-SUMOylated α-synuclein. (B) SUMOylated proteins from transfected HEK293 cells were immunoprecipitated with anti-Flag antibody (second panel), and SUMOylated α-synuclein was detected with anti–α-synuclein (first panel). IgG heavy chain is indicated. Graph depicts the percentage of SUMOylated α-synuclein in cells normalized to non-SUMOylated α-synuclein. (C) SH-SY5Y cells were transfected with HA–α-synuclein (wild type, A30P, A53T, and E46K disease mutations) and Flag-SUMO1, in the presence of myc-FKBP12 or myc-PIAS2. Cells were treated with proteinase K before fixation and processed for immunocytochemistry with anti-HA antibody. (Scale bar, 25 μm.) Arrows in B′ indicate cells with aggregates. (D) SH-SY5Y cells were transfected, processed, and analyzed as in C. Graph represents the percentage of cells with HA–α-synuclein aggregates. Values represent the average ± SEM of three independent experiments. Different from WT at *P < 0.05, **P < 0.01, and ***P < 0.001 (repeated-measures one-way ANOVA with Bonferroni post hoc test).