Analysis of protein levels by Western blotting confirmed reduced levels of protein expression in #1029 clones compared PINK1 Deficiency with controls

sing the indirect labeling kit in conjunction with Cy3-dUTP and Cy5-dUTP fluorescent nucleotides. The cDNA obtained was dried and resuspended in the hybridization buffer. The amount of DNA and the labeling efficiency was evaluated with a Nanodrop spectrophotometer. Fluorescently labeled cDNAs were T0070907 chemical information combined and hybridized to yeast genomic microchips constructed in our laboratory by arraying 6014 different PCR-amplified open reading frames from S. cerevisiae. Pre-hybridization, hybridization and washing conditions were essentially as described previously. The slides were scanned with a ScanArray 4000 apparatus, and the output was analyzed using GenePix Pro 6.0 software. Spots with either a diameter smaller than 120 mm or fluorescence 18316589 intensities for Cy3 and Cy5 lower than 150 units, were not considered for further analysis. Three different sets of microarray experiments were performed. In the first set of experiments we compared the expression profiles of ptc6 mutant cells with that of wild type cells by performing two independent experiments, each in duplicate. In the second series of experiments, we compared the transcriptomic profiles of ptc1 ptc6 double mutant cells with that of wild type cells. Two independent experiments were performed, each in duplicate. For these two set of experiments we only considered for further analysis genes with data in at least two out four spots. In the last set of experiments we compared the transcriptomic profiles of WT, ptc6, and ptc1ptc6 cells in the presence and the absence of rapamycin. In this case, data from duplicate experiments were combined, and the mean was calculated. A given gene was considered to be induced or repressed when the expression ratio was higher than 2.0 or lower than 0.50, respectively. The GEPAS3.0 software, now implemented in the Babelomics tool, was used to pre-process the data. The MIPS Functional Catalogue Database, at http://mips.helmholtzmuenchen.de/proj/funcatDB/search_main_frame.html, and Gene Ontology Enrichment tool available at YeastMine , were used for the functional distribution of gene lists. Different levels of dependence on Ptc6 were defined as ��totally dependent”, ��strongly dependent”, ��weakly dependent�� and independent, according to the expression of upor down-regulated genes after rapamycin treatment in ptc6 cells in comparison with wild type cells, as previously reported. The Chromatin immunoprecipitation assays Chromatin cross-linking and immunoprecipitation were carried out based on previously described methods with several modifications. Forty ml cultures were grown up to OD660 0.60.8 on YPD medium, and cells were exposed to 200 ng/ml rapamycin for 5, 15, 30 and 45 min. Then, cells were treated with 1.1 ml of 37% formaldehyde for 15 min at 24uC and quenched by addition of 2 ml of 2.5 M glycine for 5 min at 24uC. Cells were collected by centrifugation and washed twice with 10 ml of ice-cold HBS and once with 1.5 ml of 23713790 Lysis buffer. The pellet was resuspended in 300 ml of Lysis buffer with 1 mM phenylmethylsulfonyl fluoride and complete protease inhibitor mixture. One volume of zirconia-silica 0.5 mm beads was added and cells were broken at 4uC by vigorous shaking in a Fast Prep cell breaker. The chromatin was sheared using a Bioruptor Plus UCD-300 sonication device . The cleared lysate was recovered by centrifugation at 93006g for 5 min at 4uC. For chromatin immunoprecipitation, 50 ml of Protein G-Sepharose was coupled to 2.5 mg of anti c-myc monoclonal

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