Family 5, subfamily A1 Glutamate dehydrogenase 1, mitochondrial Isoform mitochondrial of Fumarate hydratase AcetylCoA acetyltransferase VDAC1 of Voltage-dependent anion-selective channel protein 1 Aspartate aminotransferase Mn Superoxide dismutase Cytochrome b-c1 complex Rieske subunit Guanine nucleotide-binding protein G (o) subunit alpha Mn Superoxide dismutase Thioredoxin-dependent peroxide reductase Heat shock cognate 71 kDa proteinSignal transduction Antioxidant defence/detoxification dysfunction Chaperone proteins doi:10.1371/journal.pone.0049846.tProteomics of p53-Regulated Pathways in BrainFigure 2. Putative network of pathways regulated by p53KO. A model of how the lack of p53 affects biological pathways that would attenuate progression of neurodegenerative disorders. Our result potentially makes p53 a novel therapeutic target for the delay, treatment, or prevention of these diseases. doi:10.1371/journal.pone.0049846.gIntensities were normalized to total gel densities and/or densities of all valid spots on the gels. Only spots with a 1.5-fold increase or decrease in normalized spot density in those samples and a statistically significant difference based on a Student’s t-test at 95 confidence (i.e., p,0.05) were considered for MS/MS analysis.In-gel trypsin digestionIn-gel trypsin digestion of selected gel spots was performed as previously described [23]. Briefly, protein spots identified as GSK3326595 site significantly altered were excised from 2D-gels with a clean, sterilized blade and transferred to Eppendorf microcentrifuge tubes. Gel plugs were then washed with 0.1 M ammonium bicarbonate NH4HCO3) at RT for 15 min, followed by incubation with 100 acetonitrile at RT for 15 min. After solvent removal, gel plugs were dried in their respective tubes under a flow hood at RT. Plugs were incubated for 45 min in 20 ml of 20 mM DTT in 0.1 M NH4HCO3 at 56uC. The DTT/NH4HCO3 solution was then removed and replaced with 20 ml of 55 mM iodoacetate (IA) solution in 1676428 0.1 M NH4HCO3 and incubated with gentle agitation at room temperature in the dark for 30 min. Excess IA solution was removed and plugs incubated for 15 min with 200 ml of 50 mM NH4HCO3 at RT. A volume of 200 ml 24272870 of 100 acetonitrile was added to this solution and incubated for 15 min at room temperature. Solvent was removed and gel plugs were allowed to dry for 30 min at RT under a flow hood. Plugs were rehydrated with 20 ng/ml of modified trypsin (Promega, Madison, WI, USA) in 50 mM NH4HCO3 in a shaking incubator overnight at 37uC. Enough trypsin solution was added in order to completely submerge the gel plugs.sample was acquired for a total of ,2.5 min. MS/MS spectra were searched GSK2606414 web against the International Protein Index (IPI) database using SEQUEST with the following parameters: two trypsin miscleavages, fixed carbamidomethyl modification, variable methionine oxidation, parent tolerance 10 ppm, and fragment tolerance of 25 mmu or 0.01 Da. Results were filtered with the following criteria: Xcorr1.5, 2.0, 2.5, 3.0 for 1, 2, 3, and 4 charge states, respectively, Delta CN0.1, and P-value (protein and peptide) 0.01. IPI accession numbers were cross-correlated with Swiss Prot accession numbers for final protein identification.Statistical analysisAll statistical analyses were performed using a Mann-Whitney U statistical test and a two-tailed Student’s t-test. p,0,05 was considered significant for differential fold-change values. Only proteins with significant p-values from both tests were conside.Family 5, subfamily A1 Glutamate dehydrogenase 1, mitochondrial Isoform mitochondrial of Fumarate hydratase AcetylCoA acetyltransferase VDAC1 of Voltage-dependent anion-selective channel protein 1 Aspartate aminotransferase Mn Superoxide dismutase Cytochrome b-c1 complex Rieske subunit Guanine nucleotide-binding protein G (o) subunit alpha Mn Superoxide dismutase Thioredoxin-dependent peroxide reductase Heat shock cognate 71 kDa proteinSignal transduction Antioxidant defence/detoxification dysfunction Chaperone proteins doi:10.1371/journal.pone.0049846.tProteomics of p53-Regulated Pathways in BrainFigure 2. Putative network of pathways regulated by p53KO. A model of how the lack of p53 affects biological pathways that would attenuate progression of neurodegenerative disorders. Our result potentially makes p53 a novel therapeutic target for the delay, treatment, or prevention of these diseases. doi:10.1371/journal.pone.0049846.gIntensities were normalized to total gel densities and/or densities of all valid spots on the gels. Only spots with a 1.5-fold increase or decrease in normalized spot density in those samples and a statistically significant difference based on a Student’s t-test at 95 confidence (i.e., p,0.05) were considered for MS/MS analysis.In-gel trypsin digestionIn-gel trypsin digestion of selected gel spots was performed as previously described [23]. Briefly, protein spots identified as significantly altered were excised from 2D-gels with a clean, sterilized blade and transferred to Eppendorf microcentrifuge tubes. Gel plugs were then washed with 0.1 M ammonium bicarbonate NH4HCO3) at RT for 15 min, followed by incubation with 100 acetonitrile at RT for 15 min. After solvent removal, gel plugs were dried in their respective tubes under a flow hood at RT. Plugs were incubated for 45 min in 20 ml of 20 mM DTT in 0.1 M NH4HCO3 at 56uC. The DTT/NH4HCO3 solution was then removed and replaced with 20 ml of 55 mM iodoacetate (IA) solution in 1676428 0.1 M NH4HCO3 and incubated with gentle agitation at room temperature in the dark for 30 min. Excess IA solution was removed and plugs incubated for 15 min with 200 ml of 50 mM NH4HCO3 at RT. A volume of 200 ml 24272870 of 100 acetonitrile was added to this solution and incubated for 15 min at room temperature. Solvent was removed and gel plugs were allowed to dry for 30 min at RT under a flow hood. Plugs were rehydrated with 20 ng/ml of modified trypsin (Promega, Madison, WI, USA) in 50 mM NH4HCO3 in a shaking incubator overnight at 37uC. Enough trypsin solution was added in order to completely submerge the gel plugs.sample was acquired for a total of ,2.5 min. MS/MS spectra were searched against the International Protein Index (IPI) database using SEQUEST with the following parameters: two trypsin miscleavages, fixed carbamidomethyl modification, variable methionine oxidation, parent tolerance 10 ppm, and fragment tolerance of 25 mmu or 0.01 Da. Results were filtered with the following criteria: Xcorr1.5, 2.0, 2.5, 3.0 for 1, 2, 3, and 4 charge states, respectively, Delta CN0.1, and P-value (protein and peptide) 0.01. IPI accession numbers were cross-correlated with Swiss Prot accession numbers for final protein identification.Statistical analysisAll statistical analyses were performed using a Mann-Whitney U statistical test and a two-tailed Student’s t-test. p,0,05 was considered significant for differential fold-change values. Only proteins with significant p-values from both tests were conside.
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