Fig. 6

Effect of Ser129-phosphorylation on α-syn solubility change by mitochondrial complex I inhibition. Wt-aS/SH #4 cells were fractionated into 1% Triton X-100 soluble and insoluble fractions by centrifugation at 100,000×g for 30 min. 1% Triton X-100 insoluble pellets were resolved by 8 M urea / 2% SDS solution. In 1% Triton X-100 soluble fractions, the extract (2.5 μg / lane) were loaded onto SDS-PAGE. In 1% Triton X-100 insoluble fractions, samples corresponding to 15 μg of soluble fractions were loaded. These samples were analyzed by western botting with EP1536Y, Syn-1, or AC-15 antibody. a Solubility change of α-syn by rotenone treatment. Cells were incubated by 10 or 50 nM rotenone for 5 days. The representative blots are shown. b CHX-chase experiments for analyzing alteration in the metabolic fates of insoluble Ser129-phosphorylated and total α-syn by MG132 treatment. After treatment with 50 nM rotenone for 5 days, cells were incubated in fresh media further containing 100 μM cycloheximide (CHX) and either 0.1% DMSO or 10 μM MG132 until 120 min. Graphs show the metabolic fates of Ser129-phosphorylated α-syn (left) and total α-syn (right). Data show means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction (*, P < 0.05; **, P < 0.01)