Biochemical analysis of α-Syn inoculated CNS tissue. (A) Immunoblots of brainstem samples sequentially extracted with the following: high-salt buffer (HS), HS+1% Triton-X (HS+Tx), 1% SDS (SDS) and formic acid (FA). Tissue was from untreated asymptomatic (Asym) M83 mice, aged symptomatic mice (Sym), or mice injected with PBS, symptomatic lysate, or α-Syn1-120Myc PFFs for 90 d. Immunoblots were probed with anti–α-Syn antibodies including anti–C terminus (Syn211), anti-phosphorylated Ser129 (pSyn), anti–mouse α-Syn (mSyn), or anti–α/β-Syn (Syn214). GAPDH is the loading control. Immunoblots are representative of two independent extraction experiments (n = 3 brains per group). (B) Various CNS regions from M83 mice injected with either symptomatic lysate or α-Syn1-120Myc PFFs for 90 d were sequentially extracted and probed using SNL4, which recognizes the N terminus of α-Syn. Samples from aged symptomatic M83 mice or mice injected with PBS are shown as positive controls. (C) Brainstem neurons of M83 mice injected with α-Syn1-120Myc PFFs (90 d) were double-immunostained using anti-pSyn and a rabbit polyclonal antibody specific for murine α-Syn (mSyn). Arrowheads show endogenous mSyn in pathological inclusions. (D) M83 were mice sacrificed 7 dpi with α-Syn1-120Myc PFFs. Tissue was double-immunostained for MAP-2 (red) and Myc (green) to detect internalization of exogenous Myc-tagged α-Syn by neurons. Cell nuclei were stained with DAPI (blue). Arrowheads denote intracellular Myc staining in cortical and striatal neurons. (E) Sections from α-Syn1-120Myc PFF-injected animals were double-immunolabeled against Myc and pSyn. Arrowheads denote accumulation of pSyn around internalized α-Syn. Bars: 50 µm (C); 15 µm (D); 10 µm (E).
