PMCA seeding activity in a panel of brain samples from sick or healthy M83 mice. A) Immunoblots of α-syn (C-20R antibody) in PMCA products obtained after seeding C57Bl/6-WT (10%, 144 cycles) substrate with brain samples from healthy M83+/+ mice at dilution 10−3. One or 2 PMCA rounds were performed. B) Immunoblots of α-syn (C-20R antibody) in PMCA products obtained after seeding M83+/− (5%, 96 cycles) or C57Bl/6-WT (10%, 144 cycles) substrates with brain samples from sick M83+/+ mice at dilution 10−4. The figures below each of the panels indicate the reference of the mouse, as listed in Table 1. Bars to the left of each panel indicate the ∼20-, 30-, 40-, 50-, and 60-kDa MW markers. C) ELISA detection of Ser129 phosphorylated α-syn in the same panel of brain samples from healthy or sick M83+/+ mice. ELISA detection was performed using Syn303 as a capture antibody, and rabbit pAb against Ser129 phosphorylated α-syn for detection. Two quantities of brain homogenates were examined, including 0.2% [as described by Sargent et al. (11)] and 0.01% homogenates, this last concentration corresponding to a 10−4 dilution of seeds as examined in PMCA experiments. KO, knockout; Rec, human recombinant α-syn control; WT, wild type.