Journal: Journal of Neural Transmission

Article Title: A53T-alpha-synuclein-overexpression in the mouse nigrostriatal pathway leads to early increase of 14-3-3 epsilon and late increase of GFAP

doi: 10.1007/s00702-011-0717-3

Figure Lengend Snippet: Immunohistochemical validation of upregulated striatal proteins. Striatal tissue from late age animals was analyzed. 14-3-3 epsilon staining showed neuronal immunoreactivity throughout the cytoplasm in wild-type ( a) and A53T-SNCA overexpressing mice ( b ) (corpus callosum on the left side and the ventricle below). In the mutant tissue, the neuronal cytoplasmic staining was enhanced, in agreement with the proteome results, with stronger signal intensity at the cell border (see high-magnification inset). GFAP staining showed comparable astrogliosis throughout the corpus callosum of wild-type ( c ) and mutant ( d ) aged animals (corpus callosum on the right side and the ventricle below), while the astrogliosis in the striatum appeared enhanced in the A53T-SNCA overexpressing mice, again in concordance with the proteome data

Article Snippet: After washing with 0.1% Tris–HCl, sections were incubated with biotinylated goat anti-mouse (for 14-3-3 epsilon) or anti-rabbit (for GFAP) IgG (1:200; Vector Laboratories) for 90 min at 25°C.

Techniques: Immunohistochemical staining, Staining, Mutagenesis

Journal: Journal of Neural Transmission

Article Title: A53T-alpha-synuclein-overexpression in the mouse nigrostriatal pathway leads to early increase of 14-3-3 epsilon and late increase of GFAP

doi: 10.1007/s00702-011-0717-3

Figure Lengend Snippet: Proteins identified in striata of cytosolic lysates of A53T transgenic mice by nanoLC–ESI–MS/MS analysis

Article Snippet: After washing with 0.1% Tris–HCl, sections were incubated with biotinylated goat anti-mouse (for 14-3-3 epsilon) or anti-rabbit (for GFAP) IgG (1:200; Vector Laboratories) for 90 min at 25°C.

Techniques: Transgenic Assay, Sequencing, Concentration Assay, Marker, Activity Assay, Mutagenesis

Journal: Journal of Neural Transmission

Article Title: A53T-alpha-synuclein-overexpression in the mouse nigrostriatal pathway leads to early increase of 14-3-3 epsilon and late increase of GFAP

doi: 10.1007/s00702-011-0717-3

Figure Lengend Snippet: Detailed description of the expression changes of 14-3-3 proteins. a Mass spectrum of a peptide identifying 14-3-3 epsilon. Mass spectrometric analysis of the protein spot 2 (see Fig. ) unequivocally identified the epsilon isoform of 14-3-3 protein family based on the C-terminal peptide sequence EALQDVEDENQ. b Alignments of the C-termini of several 14-3-3 protein isoforms demonstrate that the peptide identified by MS/MS ( a ) corresponds to the epsilon isoform of the 14-3-3 protein family

Article Snippet: After washing with 0.1% Tris–HCl, sections were incubated with biotinylated goat anti-mouse (for 14-3-3 epsilon) or anti-rabbit (for GFAP) IgG (1:200; Vector Laboratories) for 90 min at 25°C.

Techniques: Expressing, Sequencing, Tandem Mass Spectroscopy

Journal: Journal of Neural Transmission

Article Title: A53T-alpha-synuclein-overexpression in the mouse nigrostriatal pathway leads to early increase of 14-3-3 epsilon and late increase of GFAP

doi: 10.1007/s00702-011-0717-3

Figure Lengend Snippet: Validation of elevated 14-3-3 epsilon levels. The overexpression of 14-3-3 epsilon detected in the 2D-DIGE study was validated with independent techniques in the nigrostriatal projection via immunoblotting and qPCR analyses. a Analysis of 14-3-3 epsilon protein levels in the striatum via immunoblotting confirmed increased 14-3-3 epsilon expression. Beta-actin was used as endogenous control for normalization. b Analysis of 14-3-3 epsilon transcript levels in the midbrain via qPCR showed significantly increased 14-3-3 epsilon expression, while the levels in the striatum were not increased. TATA-binding protein ( Tbp ) was used as endogenous control. c Analysis of protein levels of the different 14-3-3 isoforms in aged striatum via immunoblotting demonstrated significantly increased 14-3-3 eta levels and significantly decreased 14-3-3 gamma levels. Data sets were normalized to the corresponding WT mean values, Student’s t test was applied for statistical analyses and significant alterations were highlighted with asterisks (* p < 0.05). N = 3–8 animals/genotype, a shows the combined data derived from two independent experiments

Article Snippet: After washing with 0.1% Tris–HCl, sections were incubated with biotinylated goat anti-mouse (for 14-3-3 epsilon) or anti-rabbit (for GFAP) IgG (1:200; Vector Laboratories) for 90 min at 25°C.

Techniques: Over Expression, Western Blot, Expressing, Binding Assay, Derivative Assay