Fig. 5
From: Mechanisms underlying extensive Ser129-phosphorylation in α-synuclein aggregates
Ser129-mediated proteasomal targeting of soluble α-syn in mitochondrial complex I inhibition by rotenone. In each treatment, the concentration of DMSO was prepared to be equal. Cell lysates (2.5 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or AC-15 antibody. a CHX-chase experiments for analyzing alteration in the metabolic fates of Ser129-phosphorylated and total α-syn by MG132 treatment. Wt-aS/SH #4 cells were pre-incubated in media containing either DMSO or 10 μM rotenone for 8 h. Then, they were incubated in fresh media further containing 100 μM cycloheximide (CHX) and/or 10 μM MG132 until 120 min. b Comparison of metabolic fates of rotenone-induced Ser129-phosphorylated α-syn with physiologically phosphorylated α-syn. Cells were pre-incubated in media containing DMSO or 10 μM rotenone for 8 h before CHX-chase experiments. Representative blots are shown. In a and b, graphs show the metabolic fates of Ser129-phosphorylated α-syn (left) and total α-syn (right). Black, yellow, blue and red lines represent treatment with DMSO, rotenone, MG132 and rotenone plus MG132, respectively. Data show means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction (*, P < 0.05; **, P < 0.01)