Fig. 2

Accumulation of PFF-induced ps-αSyn in mitochondria. a PNS prepared from αSyn monomer-treated or PFF-treated primary neurons were subjected to iodixanol gradient separation. The presence of each protein was detected by immunoblot analyses. b PNS from untreated (−) or αSyn monomer (M)- or PFF (F)-treated rat primary neurons were subjected to differential centrifugation to separate the mitochondrial and cytosolic/microsomal fractions, which were verified by immunoblot analyses with antibodies against mitochondrial ATP synthase (ATP5A) and cytosolic GAPDH, respectively. The ps-αSyn in these fractions was detected by immunoblot analysis. The bar graph in the middle shows the average ± standard error of mitochondrial ps-αSyn from five independent experiments. “C” represents control. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test (F = 322.8; n = 5; p < 0.0001). The bar graph on the right shows the ratio of ps-αSyn in the mitochondria over ps-αSyn in the cytosolic/microsomal fraction, which was the average ± standard error of three independent experiments. Statistical significance was determined by paired t-test (n = 3; p = 0.048). * represents p < 0.05. c The mitochondria isolated from PFF-treated neurons were subjected to iodixanol gradient separation, and the presence of ps-αSyn and the mitochondrial marker ATPIF1 was detected by immunoblot analyses