Biochemical analysis of α-synuclein in brain tissues. a The samples were sequentially fractionated by resolubilization with increasingly stringent buffers containing Tris-buffered saline, Triton X-100, sarkosyl, and urea. The samples were analyzed using the α-synuclein antibodies LB509 and pSyn#64. In the urea-extracted fraction, a phosphorylated α-synuclein band at approximately at ~15 kDa (arrow) was substantially accumulated in the brain (amygdala and parahippocampus) of the autopsied patient. Conversely, the level of α-synuclein in the Tris- and Triton X-100-soluble fractions was decreased. Anti-β-actin immunoblotting served as a loading control (bottom panel). b α-Synuclein-reactive fragments of dimers migrating at approximately ~30 kDa (arrowhead) and a smear at higher molecular weights were abundant in the urea-extracted fraction of the patient’s brain.