Biochemical and immunoelectron microscopy characterization of
mitochondrial α-synuclein in human PD and normal brains. Western
blot analysis of 50 μg of mitochondrial fraction from substantia nigra of
PD #1 for α-synuclein (A), mitochondrial (mt)HSP70
(B), CcO (C), and TOM20 (D) following the treatment
with trypsin, digitonin, and sodium carbonate. Sup = supernatant,
Dig = digitonin. E, Western blot analysis of 50 μg of
cytosol from substantia nigra of PD #1 treated with or without trypsin (30
μg/mg of protein) using antibodies to α-synuclein. Immunoelectron
microscopy analysis of α-synuclein in the mitochondria of substantia
nigra of PD subject (PDS #1) (F) and normal subject (NS #11)
(G) using anti rabbit antibodies toα-synuclein. The sections
were stained with secondary antibody conjugated to 20 nm (E) and 10
nm (F), respectively. M = mitochondrion, V =
vesicle, C = cytosol. Bar = 100 nm. Arrows indicate
association of gold particle to the outer membrane of mitochondria.