Fig. 4

Proteomic analysis in the insoluble fraction of α-syn PFF treated M83 neurons. A M83 neurons treated with α-syn PFFs or PBS for 14± 1 and 21± 1 days were sequentially extracted with 1% Triton X-100 followed by 2% SDS. Western blot analysis showed significant enrichment of pS129 α-syn in the Triton X-100 insoluble fraction from PFF treated neuron samples. With PBS treatment, α-syn was extracted in 1% Triton X-100 fraction. B Insoluble fractions analyzed by Mass spectrometry identified 92 proteins that were changed in the total proteome with PFF treatment at logFC cutoff of ≥ 0.25. An enrichment analysis using metascape for these 92 proteins was performed, which identified membrane/vesicle trafficking, autophagy, endocytosis, and exocytosis mechanisms. C Based on a stringent cutoff of 0.999 in CPDB, a first order network of these 92 proteins including 1640 proteins and 4,738 interactors was generated. Based on this, a zero-order network was established using the internally developed network-based visual analytics tool to identify enriched pathways which are shown in the figure. The most enriched pathways in this network included the mitochondrial pathway, translation, ubiquitin related, lysosome, unfolded protein response, vesicle trafficking and mRNA splicing