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Fig. 1 Identification of a novel phosphorylation site of human α-synuclein at T64. (A) α-synuclein of N. furzeri or O. latipes brains were immunoprecipitated and subjected to liquid chromatography–tandem mass spectrometry. The figure indicates phosphorylation sites identified in N. furzeri α-synuclein or O. latipes α-synuclein through tolerant screening. Identified residues are indicated by bold red characters. Please also refer SI Appendix, Table S1. (B) α-synuclein of human brains (nucleus basalis of Meynert) were immunoprecipitated and subjected to liquid chromatography–tandem mass spectrometry. The figure indicates comparison of the phosphorylation sites identified in human, N. furzeri and medaka α-synuclein through tolerant screening. Red squares show the specific phosphorylation site of N. furzeri α-synuclein, blue squares show the T64 phosphorylation site of human α-synuclein, and green squares show mutation sites in human familial PD. Patients’ profiles are described in SI Appendix, Table S3. Please also refer SI Appendix, Table S1. (C) Validation of phosphorylated N. furzeri α-synuclein antibodies using CIP treatment or α-synuclein KO samples. CIP+: N. furzeri Brain lysates with CIP treatment. CIP-: N. furzeri Brain lysates without CIP treatment. WT: Brain lysates of wild-type N. furzeri. KO: Brain lysates of α-synuclein KO N. furzeri. (D) Dot blotting analysis showed a highly specific reaction of anti-human T64-P α-synuclein antibody. Each synthetic peptide corresponding to the amino acids of human α-synuclein T33-P, T44-P, T54-P, T64 and T64-P was applied to a hydrophilized PVDF membrane. (E) The western blotting shows the analysis of N. furzeri brains (TBS-insoluble SDS-soluble fraction) at 1 and 5 mo using anti-S60-P N. furzeri α-synuclein. The red arrow indicated S60 phosphorylation. The graph shows quantitative analysis of S60 phosphorylation in SDS-soluble fraction using N = 4 samples/group. *P = 0.0358. (F) The western blotting shows the analysis of human PD brains (amygdala, TBS-insoluble SDS-soluble fraction) using anti-T64-P human α-synuclein. The red arrow indicated T64 phosphorylation. C: control, PD: Parkinson’s disease. The graph shows quantitative analysis of T64 phosphorylation in SDS-soluble fraction using N = 4 samples/group. *P = 0.0452. Patients’ profiles are described in SI Appendix, Table S3.