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Et al. 2011; Van Laar et al. 2011). Subsequent studies,2013 The Authors Genes to Cells 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty LtdPINK1 and Phospholipase site Parkin in major neuronshowever, by two distinctive groups along with us have successfully demonstrated the translocation event [(Cai et al. 2012; Joselin et al. 2012) and this work]. We suggest that methodological variations likely account for the seemingly conflicting observations. The study by Sterky et al. employed adeno-associated virus encoding mCherry-Parkin that was delivered by stereotactic injections to midbrain dopaminergic neurons of Tfam-loss mice (MitoPark mice; genotype TfamloxP/loxP; DAT-cre; ROSA26+/lox-Stop-lox-mito-YFP) (Sterky et al. 2011), although Van Laar et al. (2011) employed Lipofectamine 2000 to transfect wild-type rat key cortical neurons with human Parkin. In contrast, we applied main neurons derived from PARKINmice infected with a lentivirus encoding GFP-Parkin to examine translocation of Parkin to broken mitochondria. It really is achievable that the respective transfection efficiencies varied or that the methodological variations affected the neuronal cellular conditions, which might have impaired the behavior of exogenous Parkin. Alternatively, the presence of endogenous neuronal Parkin might account for the discrepancies. During our immunofluorescence experiments, we determined that mitochondrial localization of GFP-Parkin was much more robust in PARKINneurons than wild-type (PARKIN+/+) neurons (F.K. and N.M., unpublished information), suggesting that endogenous Parkin is additional effectively translocated by the cellular machinery to depolarized mitochondria than exogenous Parkin. Intriguingly, each the E3 activity and translocation of Parkin toward depolarized mitochondria have been attenuated by diseaserelevant Parkin mutations in principal neurons (Fig. 3). These results underscore the relevance of mitochondrial high quality control mediated by PINK1/Parkin in neurons and shed light around the mechanism by which pathogenic mutations of PINK1 and Parkin predispose to Parkinsonism in vivo.Key neuron cultureMouse research had been approved by the Animal Care and Use Committee of Tokyo PAK manufacturer Metropolitan Institute of Medical Science. Mouse fetal brains had been taken from C57BL/6 wild-type or PARKINmouse embryos at E15-16. After removing meninges, brain tissue was dissociated into a single-cell suspension using a Sumilon dissociation answer (Sumitomo Bakelite, Japan). Cells were plated at a density of three 9 105 cells/ mL on poly-L-lysine (Sigma)-coated dishes with the medium containing 0.339 Sumilon nerve-culture medium (Sumitomo Bakelite), 0.67 FBS (Equitech-bio, USA), 0.679 neurobasal medium, 0.679 B27 supplements, 0.679 Glutamax (above 3 reagents are from Life Technologies) and 0.67 PenStrep. 3 days following plating (at day 4), neurons had been infected with lentivirus containing HA-PARKIN, GFP-PARKIN or PINK1-Flag. Following four h of infection, the virus medium was removed. Neurons were treated with CCCP (30 lM) for 1 h at day 7 after which harvested for immunoblotting or subjected to immunocytochemistry.Traditional and phos-tag immunoblottingTo detect ubiquitylation and phosphorylation, lysates of mouse major neurons have been collected in TNE-N+ buffer [150 mM NaCl, 20 mM Tris Cl (pH 8.0), 1 mM EDTA and 1 NP-40] in the presence of 10 mM N-ethylmaleimide (Wako chemicals) to safeguard ubiquitylated proteins from deubiquitylase and phosSTOP (Roche) to shield phosphorylated proteins from.