Biochemicals)]. After centrifugation at 15,000 g for 15 minutes at 4°C, whole cell lysate was collected from the resulting supernatant and protein content determined using the Bradford method [2]. Mito- chondrial fractions or whole cell protein samples were run on 4–12% or 10% NuPAGE® Bis-Tris Gels (Invitrogen) followed by the transfer of protein to polyvinyl diﬂuoride membranes (Immobilon). Mem- branes were blocked with a 5% blocking solution (5% powdered milk dissolved in 0.1% Tween/phosphate buffered saline) prior to incubation with 3X-FLAG (1:1000 dilution; Sigma Aldrich), parkin (1:1000 dilution; Millipore), or ubiquitin (1:1000 dilution; Dako Cytomation) antibodies; Negative reaction con- trols included incubation of tissues in the absence of primary antibody. β-Actin (1:5000 dilution; Milli- pore) or VDAC1 (1:1000 dilution; Abcam) antibodies were used as whole cell or mitochondrial protein loading controls, respectively. Membranes were incu- bated in corresponding HRP-conjugated secondary antibodies (Santa Cruz) for 1 hour following incuba- tion with primary antibody. Protein bands were detected using chemiluminescence substrate for horse-radish peroxidase (HRP) (Amersham Biosci- ences, Millipore). Resulting bands were captured on Kodak BioMax Light ﬁlm, developed, scanned and densitometry measured and quantiﬁed using the NIH Image J application. Immunocytochemistry (ICC) To assess mitochondrial parkin and lysosomal LC3 expression, cells were seeded in 8-chamber slides (Thermo Scientiﬁc/Nunc), treated, incubated with 10 μM Mitotracker Red® (Invitrogen) for 20 minutes, and then ﬁxed with 4% paraformaldehyde solution (PFA) for 20 minutes. Once ﬁxed, cells were washed twice with PBS. Fixed cells were then blocked with 5% normal donkey serum (NDS) for 1 hour at room tem- perature followed by an overnight incubation with pri- mary antibodies (parkin or LC3) at 4°C. Cells were then incubated with Alexa-conjugated ﬂuorescent sec- ondary antibodies (Invitrogen) for 1 hour in the absence of light at room temperature to allow the ﬂuorescent detection of desired proteins. Images were captured using a Zeiss LSM 510 confocal microscope. Mitochondrial respiration assays via the XF24 Seahorse microrespirometry For these experiments, cells were seeded onto 24-well Seahorse microplates (40,000 cells per well) prior to analysis in the Seahorse XF24. All experiments were conducted at 37°C. Wells containing an estimated volume of 500 μl of Seahorse media (3.5 mM KCl, 120 mM NaCl, 1.3 mM CaCl2-2H2O, 0.4 mM KH2PO4, 1.2 mM Na2SO4, 15 mM D-Glucose, Rapamycin reverses effects of parkin activity loss 1013 2 mM MgSO4 and 10 mM TES) were analysed for mean oxygen consumption rates (basal respiration, following addition of 4 μg/ml oligomycin to assess proton leak, and 6 μm FCCP for maximal respira- tion). The GraphPad Prism program was used for statistical analysis of respiration measurements using the Tukey–Kramer method. Mitochondrial complex I activity Complex I activity was assayed in isolated mitochon- drial fractions as rotenone-sensitive NADH dehydro- genase activity as previously described [24]. Values were normalized per protein using Bradford Assay. Statistics Results One-way ANOVA followed by Newman–Keuls Mul- tiple comparison was used to compare differences between groups unless otherwise noted. MAO-B induction results in decreases in parkin’s ubiquitin E3 ligase activity We ﬁrst assessed whether MAO-B elevation had effects on parkin E3 ligase function that could pre- vent the enzyme from ubiquitin-tagging dysfunc- tional mitochondria for lysosomal degradation. To monitor E3 ligase activity, we assessed parkin auto- ubiquitination in dox-inducible MAO-B cells stably transfected with human parkin in the absence and presence of dox or FCCP-induced mitochondrial depolarization (Figure 1). MAO-B-induced oxidative stress was found to negatively impact on parkin’s ability to ubiquitinate itself. Mitochondrial depolar- ization via FCCP in contrast was not found to inhibit E3 ligase activity. Parkin’s translocation to mitochondria increases in response to MAO-B induction and is exacerbated in cells treated with FCCP and/or rapamycin Parkin recruitment to damaged mitochondria has been reported to increase in response to depolariza- tion by FCCP (31). Direct oxidative damage of mito- chondria (such as that induced by MAO-B increase) may also result in their depolarization and subsequent parkin recruitment. In the present study, immunoblot analysis of whole mitochondrial fractions isolated from MAO-B-parkin cells revealed increased mito- chondrial parkin levels in response to both MAO- B-induced oxidative stress and depolarization via FCCP and in combination were found to be additive (Figure 2). Rapamycin treatment in the context of either MAO-B induction or depolarization also