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70 8 2011 05 31 MOCHIZUKI 200768 SDC3

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70 8 2011 05 31 MOCHIZUKI 200768 SDC3

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  1. SupplementalFigure 3. Effects of overexpressed parkin on mitochondrial alterations. A:Western blotting was performed using midbrain tissues of mice sacrificed 7 dafter implantation of minipumps. PINK1 protein level was slightly but non-significantly increased in the parkin-overexpressed side (parkin) of saline- (saline) and MPTP-minipump mice (MPTP) as compared to the hrGFP-overexpressed sides(GFP). Another clone of anti-p-αSyn antibody (clone pSyn#64) also showed MPTP-induced increase of the p-αSyn.B–D:Percentagesof control (GFP/saline group) for PINK1/Actin (B), DJ-1/Actin (C), and Tom20/Actin (D). There were no statistically significant differences among the groups. E–J:Nigral sections of saline-minipump, MPTP-minipump (50 mg/kg/day)(28 dafter the implantation of minipumps), and MPTP sub-acute mice (30mg/kg/day for 5 consecutive d; 25 dafter the first injection of MPTP),immunostained for Tom20. The minipump-mediated MPTPtreatmentincreased Tom20 immunoreactivity in the SNpc (compare E–G, and H–J).Thealterations were not evident in sub-acute MPTP group.(K–Z’), Nigral sections of rAAV1-injected mice immunostained for Tom20 (K, O, S, and W),or co-immunostained for hrGFP (L,N,N’,P,R, and R’, shown in green in (N, N’, R, and (R’),or parkin (T,V,V’,X,Z, and Z’, (green in V, V’, Z, and Z’), and Tom20 (M–N’ Q–R’,U–V’, and Y–Z’), red in(N, N’, R, R’, V, V’, Z, and Z’)) at 28dpost-implantation of minipumps. Boxed areasin N, R, V, and Zare enlarged in N’, R’, V’, and Z’, respectively. MPTP treatment increased Tom20 immunoreactivity in the SNpc of both rAAV1-hrGFP-injectedand rAAV1-parkin-injected mice (compare KandO,Sand W, M, N’ and Q, R’, andU, V’ and Y, Z’). Parkin overexpression did not affect Tom20 immunoreactivity in MPTP mice (compare (O)and (W), and (Q,R’)and (Y, Z’)). Intracellular distribution of parkin was similar to that of hrGFP (compare (L–N’)and (T–V’), and (P–R’)and (X–Z’)); i.e.parkin did not apparentlycolocalizewith Tom20. Numbersof analyzed mice in each group are indicated within the bars. Data are mean±SEM. N.S., not significant (one-way ANOVA). Scale bar in (E), 500-μm(applicable to (E–G)); in (H), 50-μm(H–J); in (K), 50-μm ((K),(O),(S),and(W)); and in (L), 10-μm ((L–N),(P–R),(T–V), and (X–Z)).

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