FIGURE 8.

Interaction of α-synuclein with mitochondrial complex I. A, effect of in vitro incubation of mitoplast from substantia nigra of control subject #9 with dilution buffers (a) and varying concentrations of α-synuclein (b). Effect of α-synuclein (1.5 μg/mg of mitochondrial protein) on the activities of complex II (B), complex III (C), and complex IV (D). Mitochondria from striatum of a normal subject (NS) and a PD subject (PDS), as well as DAN cells expressing α-synuclein constructs were solubilized and run on blue native PAGE. Following the separation, complexes were transferred on polyvinylidene difluoride membranes and stained with antibodies to NDUFA9 (E) and α-synuclein (F). Co-immunoprecipitation of α-synuclein using complex I immunocapture kit (Mitosciences). Immunocaptured mitochondrial complexes I (4 μg) from striatum of PD and normal subjects as well as DAN cells expressing α-synuclein constructs were resolved on 14% SDS-PAGE and subjected to Western immunoblot using antibodies to α-synuclein (G) and NDUFA9 (H).