Yeast Two-Hybrid Screening
MaV203 was transformed with pDBLeu-R1-parkin, and 3 × 106
stable transformants were further transformed with 15 µg of pPC86 human brain cDNA library (Life Tech/Gibco). Transformants were selected and confirmed according to the manufacturer's instructions as previously described (Zhang et al., 2000
Polyclonal PARIS antibodies were generated as described in supplemental information. Primary antibodies used include the following: goat anti-PGC-1α (K-15, Santa Cruz Biotechnology), mouse anti-PGC-1α (4C1.3, Calbiochem), goat anti-NRF1 (A-19, Santa Cruz Biotechnology), rabbit anti-NRF1 (ab34682, Abcam), mouse anti-parkin (Park8, Cell Signaling), rabbit anti-TH (Novus Biologicals), rabbit anti-glutamate decarboxylase (GAD) 65&67 (Millipore), rabbit anti-GFP (ab661, Abcam), mouse anti-GFP (ab1218, Abcam); Secondary antibodies used include Biotin-SP-conjugated goat anti-rabbit (Jackson ImmunoResearch lab), donkey anti-goat-Cy3, donkey anti-rabbit-Cy2/Cy3, donkey anti-mouse-Cy2 for immunostaining.
Full-length parkin, and deletion mutants, Q311X, R42P, R275W, G430D and C431F parkin, HA-ubiquitin, PARIS, PARIS deletion and point mutations, GST-PARIS, ZNF398 vector were constructed as described in supplemental information. Construct integrity was verified by sequencing. Lentiviral pLV-PGC-1α plasmid was kindly provided by Dr. Dimitri Krainc (Massachusetts General Hospital, Harvard Medical School, Charlestown, USA), MYC-tagged XIAP was generously given from Dr. Kenny K. K. Chung (Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong),
Cell culture and Transfection
Human neuroblastoma SH-SY5Y cells (ATCC, Manassas, VA) were grown in DMEM containing 10% FBS and antibiotics in a humidified 5% CO2
/95% air atmosphere at 37°C. For transient transfection, cells were transfected with indicated amounts of target vector using Lipofectamine Plus (Invitrogen), according to manufacturer’s instructions. For co-immunoprecipitation from cell cultures, SH-SY5Y cells were transfected with 2 µg of each plasmid, unless otherwise indicated in supplemental information. For the ubiquitination assay, SH-SY5Y cells were transiently transfected with 2 µg of pRK5-Myc-tagged parkin, Myc-tagged parkin (C431F, G430D, R275W, Q311X) pCMV-FLAG-PARIS, and 2 µg of pMT123-HA-ubiquitin plasmids for 48 h. For the luciferase assay SH-SY5Y cells were transiently transfected with pCMV-empty vector or pCMV-FLAG-PARIS with either wild type or Q311X parkin, or pGL3-Basic, pGL3-PGC-1α promoter-Luciferase, or pGL3-PGC-1α promoter deletion mutant (a gift from Akyoshi Fukamizu, University of Tsukuba, Japan) (Daitoku et al., 2003
) for firefly Luciferase assay and 0.1 µg pRL-TK vector (Promega) for Renilla
Immunocytochemistry and Immunblot Analysis
Immunocytochemistry and immunoblot analysis was performed as described in the supplemental information.
In vitro Interaction and Ubiquitination Assays
GST-PARIS and His-Parkin were used in in vitro interaction and ubiquitination assays as described in the supplemental information.
CAST, EMSA, ChIP, qRT-PCR Assays
We followed a previous published protocol with modification for CAST ((Cyclic Amplification and Selection of Targets) (Voz et al., 2000
) as described in the supplemental information. GST, GST-PARIS, GST-C571A–PARIS were used for electrophoretic mobility shift assays (EMSA) as described in the supplemental information. Chromatin immunoprecipitation was carried out according to the manufacturer’s instruction as described in the supplemental information. Primers used for real-time pRT-PCR are listed in Table S4
Conditional parkin knockout
To generate Cre-flox conditional model of parkin knock out, a lentiviral vector expressing GFP fused Cre recombinase (Lenti-GFPCre) was stereotaxically introduced into exon 7 floxed parkin mice (parkinFlx/Flx) using the coordinates in supplemental information. Furthermore lentiviral shRNA-PARIS was co-administrated along with Lenti-GFPCre to demonstrate whether the changes in PGC-1α and NRF-1 are due to PARIS.
Quantitative data is presented as the mean ± S.E.M. Statistical significance was either assessed via an unpaired two-tailed Student’s t-test or an ANOVA test with Student-Newman-Keuls post-hoc analysis. Assessments were considered significant with a p < 0.05,.