1014 A. Siddiqui et al. Figure 1. Decreased parkin ubiquitin E3 ligase activity following MAO-B induction. Inducible MAO-B PC12 cells were stably transfected with human parkin and assessed under both control and dox-induced conditions versus following FCCP treatment. Cell lysates were prepared from cells and immunoprecipitated (IP) using Flag-tagged or parkin antibody. Samples were run in an E3 ligase activity assay followed by analysis viaWestern blot using parkin and ubiquitin antibodies; β-actin was used as loading control. Corresponding densitometric analyses are shown (n 3). Values are expressed as mean SD; ∗∗∗p 0.001 versus parkin–MAO-B cells, ∗∗p 0.01 versus MAO-B cells. appeared to result in increased mitochondrial parkin levels, which were also additive in combination. To validate whether increases in mitochondrial par- kin levels were due to increased localization of the pro- tein to this organelle, parkin-MAO-B cells were examined using ﬁxed cell imaging. Higher expression of parkin (green) within mitochondria (labelled with mitotracker red), resulting in increases in merged yel- low staining was used as a marker of mitochondrial parkin localization. Mitochondrial parkin localization was found to increase in response to MAO-B induction and to be further exacerbated in the presence of FCCP, correlating with the immunoblot data (Figure 3). Rapamycin also appeared to result in increased mito- chondrial parkin protein levels; elevations in overall cel- lular parkin also appear to occur in response to either MAO-B induction or rapamycin treatment (Figure 4). Mitophagy is inhibited in response to MAO-B induction but restored in the presence of rapamycin treatment Although increased MAO-B induction appears to result in increased translocation of parkin to the mitochondria, the protein may not be functionally Figure 2. Mitochondrial parkin levels in various conditions. Western blot analyses were performed on mitochondrial sub-fractions from cells in various treatment conditions using Flag or parkin antibodies (Sigma). Immunoblots were normalized for VDAC (Chemicon) for corresponding densitometric analyses (n 3). Values are expressed as mean SD, ∗∗p 0.01 ∗∗∗p 0.001 versus MAO-B cells.