Figure 4.

Depletion of endogenous PINK1 reduces parkin phosphorylation in dopaminergic cells. SH-SY5Y cells were transfected with Myc-tagged parkin and vehicle (CTL), non-targeting (NT) siRNA or PINK1 siRNA and treated with 20 nm rotenone for 8 h as indicated. (A) The levels of PINK1, parkin and actin in the cell lysates were analyzed by immunoblotting with anti-PINK1, anti-Myc and anti-actin antibodies. (B) In vivo parkin phosphorylation was determined by immunoprecipitated with anti-Myc antibody followed by immunoblotting using anti-phosphoserine and anti-parkin antibodies. (C) Normalized levels of in vivo parkin phosphorylation by PINK1. Data represent mean ± SEM from three independent experiments. *Significantly different from the corresponding rotenone-treated controls (P < 0.05). AU, arbitrary units.