Phosphatase activity. To detect phosphorylated proteins by Page, 7.5 polyacrylamide gels containing 50 lM phos-tag acrylamide (Wako chemical substances) and one hundred lM MnCl2 were used. Following electrophoresis, phos-tag acrylamide gels had been washed with transfer buffer containing 0.01 SDS and 1 mM EDTA for ten min with gentle shaking after which washed with transfer buffer containing 0.01 SDS with out EDTA for 10 min as outlined by the manufacturer’s protocol. Proteins were transferred to polyvinylidene difluoride membranes and analyzed by standard immunoblotting. Image contrast and brightness had been adjusted in Photoshop (Adobe).Experimental proceduresLentivirusHA-PARKIN, GFP-PARKIN or PINK1-Flag genes had been cloned into a lentiviral vector (pLenti-CMV puro DEST, a kind present from Dr. Eric Campeau at Resverlogix Corp.). Lentivirus was ready following Campeau’s protocols (Campeau et al. 2009). Briefly, lentiviral particles have been developed in HEK293T cells by transfection with the aforementioned lentiviral vectors working with Lipofectamine 2000 (Life Technologies). A lentivirus-containing supernatant was collected 48 h just after transfection and concentrated to 109 by ultracentrifugation at 37,000 9 g for 2 h.ImmunocytochemistryPrimary neuron cells have been fixed with four paraformaldehyde, permeabilized with 50 lg/mL digitonin and stained with primary antibodies described under and together with the following secondary antibodies: mouse and rabbit Alexa Fluor 568 and 647 (Life Technologies). Neurons have been imaged utilizing a laser scanning microscope (LSM780; Carl Zeiss, Inc.).AntibodiesAntibodies applied in this study are as follows: Cyclin G-associated Kinase (GAK) Inhibitor web anti-Tom20 (FL145; Santa Cruz Biotech.), anti-Parkin (PRK8; Sigma),2013 The Authors Genes to Cells 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty LtdGenes to Cells (2013) 18, 672F Koyano et al. anti-Tom70 (gift from Dr. Otera), anti-b-Tubulin isotype 3 (SDL.3D10; Sigma), anti-Miro1 (RHOT1; Sigma), anti-Mitofusin2 (ab56889; Abcam), anti-VDAC1 (ab-2; Calbiochem), anti-PINK1 (BC100-494; Novus) and anti-HKI (C35C4; Cell Signaling) antibodies. are ubiquitinated within a PINK1/parkin-dependent manner upon induction of mitophagy. Hum. Mol. Genet. 19, 48614870. Geisler, S., Holmstrom, K.M., Skujat, D., Fiesel, F.C., Rothfuss, O.C., Kahle, P.J. Springer, W. (2010) PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/ SQSTM1. Nat. Cell Biol. 12, 11931. HCV Protease Species Glauser, L., Sonnay, S., Stafa, K. Moore, D.J. (2011) Parkin promotes the ubiquitination and degradation from the mitochondrial fusion factor mitofusin 1. J. Neurochem. 118, 636645. Imaizumi, Y., Okada, Y., Akamatsu, W., et al. (2012) Mitochondrial dysfunction linked with increased oxidative tension and alpha-synuclein accumulation in PARK2 iPSCderived neurons and postmortem brain tissue. Mol. Brain five, 35. Jin, S.M., Lazarou, M., Wang, C., Kane, L.A., Narendra, D.P. Youle, R.J. (2010) Mitochondrial membrane prospective regulates PINK1 import and proteolytic destabilization by PARL. J. Cell Biol. 191, 93342. Joselin, A.P., Hewitt, S.J., Callaghan, S.M., Kim, R.H., Chung, Y.H., Mak, T.W., Shen, J., Slack, R.S. Park, D.S. (2012) ROS-dependent regulation of Parkin and DJ-1 localization for the duration of oxidative pressure in neurons. Hum. Mol. Genet. 21, 4888903. Kinoshita, E., Kinoshita-Kikuta, E. Koike, T. (2012) Phostag SDS-PAGE systems for phosphorylation profiling of proteins with a wide selection of molecular masses below neutral pH conditions. Proteomics 12, 19202. Kinoshita, E., Kinoshita-Kikut.