soluble fractions) were collected. The pellets were extracted twice with 200 ml of TBS containing 5% SDS, and the resulting supernatants were collected and pooled as the TBS-insoluble (SDS-soluble) fraction. Protein concentrations were determined using the bicucullinic acid (BCA) protein assay (Pierce). To confirm genotypes (Figure 1), TBS-soluble brain fractions (10 mg) from non symptomatic 17 months-old mice were analyzed. TBS- and SDS-soluble brain fractions (30 mg) from 17 months-old non symptomatic and 17–25 months-old symptomatic mice were used for assaying a-synuclein and PS129-a-synuclein proteins (Figure 7). GST pull-down assays were done as described previously [26], with 500 mg TBS-soluble brain fraction from a symptomatic mouse, and 5 mg of GST or GST-Parkin immobilized on glutathion-sepharose beads (Amersham Biosciences). Ubiquityla- tion assays were carried out as described in [26]. Recombinant normal a-synuclein and the S87A and S129A variants were produced, purified and phosphorylated in vitro as described previously [42]. Proteins were resolved by SDS-PAGE on 4–12% gradient gels (Invitrogen). The proteins were electrotransferred onto nitrocellu- lose membranes and probed with: monoclonal anti-Parkin (MAB5512, clone PRK8, Chemicon), anti PS129-a-synuclein (clone PSyn#64, WAKO; clone EP1536Y, Abcam), anti-a-tubulin (clone DM 1A, Sigma), anti-FLAG (M2, Sigma); polyclonal anti-a- synuclein (SA3400, Affiniti) and anti-actin (A2066, Sigma), anti- GST (GE Healthcare/Amersham). Proteins were visualized by enhanced chemioluminescence (Pierce) using a Kodak Image Station 4000 MM. The Image J program (http://rsb.info.nih. gov/ij/) was used for signal quantification. Immunohistochemistry Mice were anaesthetized with pentobarbital (130 mg/kg, i.p.; Sigma, St Quentin, France) and perfused transcardially with 4% paraformaldehyde freshly prepared in 0.1 M PBS, pH 7.4. Brains were removed and post-fixed for two hours in the same solution. Left-half brains were dehydrated by four 10 min incubations and two 20 min incubations in ethanol at 35uC, followed by three incubations of 45, 60 and 75 min each in xylene, at 35uC. The tissues were then embedded in paraffin by three incubations of 40, 15 and 15 min each at 65uC (Shandon Excelsior tissue processor from Thermo Scientific) and cut on a microtome into 5 mm-thick sections collected on slides and stored at room temperature. For immunohistochemistry, sections were deparaffinized in xylene, and hydrated in a descending series of ethanol concentrations. Antigens were retrieved by boiling the sections in 0.01 M citrate buffer, pH 6.0, five times for 3 min in a microwave at 350 Watts. For immunoperoxidase staining, sections (5 and 20 mm thick) were incubated in PBS containing 3% H2O2 and 20% ethanol for 5 min, to inhibit endogenous peroxidases. Sections were permea- bilized and non specific epitopes were blocked by incubation for 45 min in PBS containing: 2% BSA, 0.1% Triton X-100 (tyrosine- hydroxylase (TH)-staining); 2% NGS, 5% BSA, 0.1% Triton X- 100 (PS129-a-synuclein staining); or 20% newborn goat serum, 10% BSA, 0.1% Triton X-100 (ubiquitin staining and double immunofluorescent labelling). Sections were hybridized overnight at 4uC with the primary antibodies, then treated with biotinylated secondary anti-rabbit IgG antibody (Vectastain; Vector Labora- tories, Burlingame, CA, USA), and incubated with avidin and biotinylated horseradish peroxidase complex. The signal was revealed by incubating the sections in 0.25 M Tris-HCl, pH 7.4 containing 0.5% of the peroxidase substrate, 3,39-diaminobenzi- dine tetrahydrochloride, and 0.015% H2O2. The sections were dehydrated in an ascending series of ethanol concentrations, cleared in xylene, and mounted with Eukitt Mounting Medium. PARK2 Modifies Synucleinopathy For immunofluorescent staining, the sections were then incubated with the primary antibody overnight, at 4uC and the signal revealed by incubation with Alexa Fluor 488-coupled goat anti-rabbit IgG antibody (1:2000, Invitrogen) or Cy3-coupled donkey anti-mouse IgG antibody (1:200, Jackson), for one hour at room temperature. The same procedure was used twice sequentially for double stainings. Fluorescent images were acquired with an epifluorescensce microscope (Axioplan 2, Zeiss, Germany) equipped with the FluoUp image analysis system (Explora Nova, La Rochelle, France). Primary antibodies were: rabbit polyclonal anti-PS129-a-synu- clein (1:500, kindly provided by T. Iwatsubo [1]); anti-ubiquitin (1:1500, DAKO); anti-synphilin-1 (1:150, kindly provided by Simone Engelender); and anti-activated caspase 9 (1:200, Cell Signalling); rabbit monoclonal anti-PS129-a-synuclein (1:5000, Abcam); mouse monoclonal anti-PS129-a-synuclein (1:1500, WAKO); and anti-ubiquitin (1:500, Chemicon). Anti-PS87-a- synuclein antibodies were raised in rabbits against a peptide identical to residues 81–93 of human a-synuclein, with serine 87 being phosphorylated (Eurogentec), and used at a 1:100 dilution. Immunostainings for total human a-synuclein and TH were done as described in Materials and Methods S1. Quantitative immunohistochemical analyses The density of PS129-a-synuclein immunostaining was evaluated in symptomatic mice (n = 5 per genotype) by determining the mean of eight 5 mm-thick coronal hemi-brain, blind-coded sections, 120 mm apart, covering a brainstem region between bregma 22.5 and 23.5 mm, with a 256objective and using the Mercator workstation. The borders of the brainstem area on each section were drawn manually and a grid with counting frames of 1506150 mm was superimposed onto the region of interest. The number of intersections between immunolabelled structures and grid lines was registered for each section. The mean staining density was defined as the average number of intersections per section. TH-positive neurons in the substantia nigra and the locus coeruleus were quantified as described in Materials and Methods S1 and in Reference S1. Colocalization between PS129-a-synuclein and ubiquitin was analyzed in spinal cord sections processed for double immunoflu- orescent staining. Images of at least five double-immunostained neurites and five cell bodies from three animals per genotype were taken with a confocal microscope (Leica SP2 AOBS). Colocaliza- tion was evaluated with the Pearson’s coefficient calculated with the ‘‘Colocalization Threshold’’ plugin of the Java image processing program Image J (http://rsb.info.nih.gov/ij/). The proportion of PS129-a-synuclein-positive cell bodies and neurites immunopositive for ubiquitin was determined from one or two representative double immunofluorescence-labelled, 5 mm-thick brainstem sections from symptomatic mice, chosen between bregma 22.5 and 23.5 mm according to the abundance of immunolabelled structures. To determine the proportion of PS129- a-synuclein-positive neurites co-stained with anti-ubiquitin anti- bodies, fluorescent images were acquired with an epifuorescence microscope (Axioplan 2, Zeiss, Germany), with a filter set for Alexa Fluor 488 (PS129-a-synuclein) and a filter set for Cy3 (ubiquitin), and a 636objective; 30 to 130 PS129-a-synuclein- positive neurites were scored per animal (n = 5 per genotype). To determine the proportion of double-labelled cell bodies, brain sections were examined directly with the epifluorescent micro- scope and the 636objective; 30 to 200 PS129-a-synuclein-positive cell bodies were scored per animal (n = 4 per genotype). The percentage of PS129-a-synuclein- or ubiquitin-positive neurons double stained for cleaved caspase 9, and the percentage PLoS ONE | www.plosone.org 11 August 2009 | Volume 4 | Issue 8 | e6629