Fly Longevity and Oxidative Stress Assays Longevity assays were performed as previously described [81]. For oxidative stress assays a minimum of 20 male flies (2–3 days of age) was kept on filter papers soaked with paraquat (20 mM paraquat dichloride in 5% sucrose). Survival of flies was scored on a daily base. Fresh paraquat/sucrose solution was supplied daily. Negative Geotaxis Assay Fly climbing was assessed in accordance with previously published protocols [21,43,82]. Flies were aged on normal yeast medium. At ages 1 and 4 weeks, climbing was assessed (20 flies per genotype). Flies were individually placed in a graduated cylinder, and allowed to climb for 15 s. Maximum height attained was recorded, and analysis was repeated 3 times per time point, with 3 trials at one minute intervals recorded at each time point. Immunohistochemistry Fly brains were dissected in cold PBS, washed in a PBS/0.1% Triton X (PBT), fixed in 4% PFA (30 min, 4uC), and blocked in PBT containing 5% normal goat serum (overnight, 4uC). For TH staining, brains were incubated with primary anti-TH antibody (1:100; rabbit polyclonal, AB152, Chemicon International/Milli- pore) for 2 days, 4uC, and subsequently with fluorescent secondary anti-rabbit antibody (1:200; AlexaFluor-555 or Cy3; Invitrogen/ Jackson Immunological Research) for 3.5 hours. Afterwards, brains were mounted in Vectashield (Vector Labs). The number of TH-positive neurons was determined on Z-stacked confocal sections (1 mm, Leica DM IRE2, Laser) [83]. At least 15 brains were analyzed per genotype. Protein Collection and Western Blotting Fly heads were homogenized in RIPA buffer (50 mM Tris, pH 8.0, 0.15 M NaCl, 0.1% SDS, 1.0% NP-40, 0.5% Na- Deoxycholate, 2 mM EDTA, Complete Protease Inhibitors (Roche Applied Sciences), pH 7.4), centrifuged, and the superna- tant was collected. Cell culture protein samples were collected after washing cells in ice cold PBS, followed by lysis in RIPA buffer for 30 min on ice. Cell debris was removed by centrifugation, and supernatants were collected. For Western blot analysis, protein samples were separated via SDS-PAGE gel and then transferred onto nitrocellulose mem- brane. Blocking in skim milk was followed by overnight primary antibody incubation. The primary antibodies used were as follows: mouse anti-a-Synuclein (1:1000; Cell Signaling); mouse anti-Drosophila Syntaxin (1:2000 Developmental Studies Hybrid- oma Bank (DSHB)), mouse anti-b-Tubulin (1:500, DSHB); mouse anti-D Tubulin (1:10,000; Sigma-Aldrich); mouse anti-TRAP1 (1:1000; BD Biosystems); mouse anti-phospho-tyrosine (PY99) (1:200; Santa Cruz Biotechnology); mouse anti-phospho-threo- nine (H2) (1:200: Santa Cruz Biotechnology); mouse anti- phospho-serine (16B4) (1:200; Santa Cruz Biotechnology); mouse anti-Cytochrome c (1:500; Santa Cruz Biotechnology); rabbit anti-VDAC1 (0.3 mg/ml; Abcam); mouse anti-COX IV (2 mg/ ml; Abcam); rabbit anti-GFP polyclonal (1:1000; Santa Cruz Biotechnology). Appropriate secondary anti-mouse or rabbit horseradish peroxidase-linked antibodies (1:10,000) were obtained from GE Healthcare. Membranes were incubated with the secondary antibody for one hour, followed by signal detection using the Chemiglow substrate (Biozym). TRAP1 Mitigates a-Synuclein Toxicity Total RNA Isolation, cDNA Preparation, and Real-Time PCR Method for fly head RNA isolation was adapted from the following link: http://www.ou.edu/journals/dis/DIS84/Tec2% 20Bertucci/Bertucci.htm. 20 fly heads per tube were used for RNA isolation. RNA samples were treated with DNase following manufacturer’s instructions (Promega RQ1 RNase-Free DNase kit). Total RNA from cultured cells was prepared from cells using Qiagen RNeasy Mini kit (Qiagen). RNA was used for cDNA production via reverse transcription using the iScript cDNA Synthesis Kit (BioRad). Real-time PCR measurements were performed using the SYBR Green (Thermo Fisher Scientific) reagent following manufacturer’s instructions for preparation of PCR samples. Gene of interest signal was compared to that of control gene expression (b-Actin5c for fly samples and 18S for human samples) using the 22DDCt method [84]. No-RT controls were performed to exclude for genomic DNA sample contamina- tion. PCR reactions were followed by generation of a dissociation curve to check for side product generation. Amplification conditions for fly samples were as follows: 5 min at 95uC, 40 cycles of: 30 s at 95uC, 30 s at 58uC, 60 s at 72uC, followed by 10 min at 72uC. Gene of interest was normalized to control b-Actin5c signal. The primers used are listed in Table S5. Amplification conditions for cell culture samples were as follows: 5 min at 95uC, 40 cycles of: 30 s at 94uC, 30 s at 60uC, 60 s at 72uC, followed by 10 min at 72uC. Gene of interest was normalized to control 18S signal. For primers, see Table S5. Cell Culture Cloning and in vitro mutagenesis. Full length human a- Synuclein cDNA (423 bp), carrying the [A53T] mutation (cDNA a gift from Dr. Felipe Opazo, European Neuroscience Institute, Go¨ ttingen, Germany) was subcloned into the pcDNA3.1+ expression vector (Invitrogen) using HindIII and EcoRV restriction sites. Full length human TRAP1 cDNA (2115 bp) was amplified from human HEK293 cell cDNA samples. BglII and XhoI restriction sites were introduced using primers listed in Table S5. TRAP1 cDNA was then cloned into the pcDNA3.1+ vector using the BglII, XhoI sites. In vitro mutagenesis of human TRAP1 in pcDNA3.1+ was carried out using the QuickChange Site-Directed Mutagenesis kit (Stratagene) following manufacturer’s instructions. Sites for mutagenesis were based on conserved sites found in the ATPase domain (see Figure S6 for sequence homology). PCR cycling parameters were used as suggested by Stratagene, with a specific extension time of 8 min and 16 cycles for all reactions. Primers used for generating the mutants are listed in Table S5. Lentivirus preparation. Full length human TRAP1 or [A53T]a-Synuclein cDNA was subcloned into a third generation lentiviral vector pRRLsin.cPPT.PGK/GFP.WPRE (Tronolab), excluding the GFP cassette. The GFP-expressing virus served as a control [85]. Cells, plasmid transfection, and viral infection. Human HEK293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. Transfection of plasmids and small interfering RNA (siRNA) into HEK293 cells was completed using Metafectene (Biontex) following manufacturer’s instructions. siRNAs used for gene knockdown experiments were obtained from Qiagen: MAPK1 control siRNA (Qiagen 1027277); 2 different TRAP1 siRNAs (Flexitube siRNA SI03066364 and siRNA SI00115150); a scrambled siRNA for control (Allstars Negative Control, 1027280). Final concentration of siRNA used PLoS Genetics | www.plosgenetics.org 11 February 2012 | Volume 8 | Issue 2 | e1002488