Computational protocol: Altered distribution of the EphA4 kinase in hippocampal brain tissue of patients with Alzheimer’s disease correlates with pathology

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[…] Frozen hippocampal tissue slides of patients in all Braak stages (I-VI, n = 29) were cut and lysed with M-PER (pH 7.6, Thermo Scientific) containing protease and phosphatase inhibitors (Roche). After incubation for 30 min on ice and subsequent centrifugation (2 × 10 min, 4°C, 15682 × g), protein concentrations of the supernatants were determined with the standard Bradford Lowry Assay (Bio-Rad Protein Assay).Protein lysates were re-suspended in sample buffer (Thermo Scientific) and heated for 5 minutes at 95°C. Proteins were resolved by SDS-PAGE using 8–16% gradient polyacrylamide gels (mini-PROTEAN®TGX™, Bio-Rad) in running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3, Bio-Rad) and electrophoretically transferred onto a nitrocellulose membrane (0.2 μm; Whatman, Protran™) in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, Bio-Rad). Membranes were blocked for 1 hour in Tris-buffered saline (50 mM Tris pH 7.5, 0.15 M NaCl, 0.1% Tween-20) containing 5% BSA (Roche Diagnostics) and incubated over night with primary anti-EphA4 antibody (1:500). A mouse monoclonal antibody directed at the extracellular (c-terminal) domain of the Ephrin-A4 receptor (amino acids 379–472; BD Transduction Laboratories) and a rabbit polyclonal EphA4 antibody directed at the intracellular domain (amino acids 890–904; Abcam) were utilised. Incubation with a secondary antibody linked to horseradish peroxidase ([HRP]-anti-rabbit IgG or HRP-anti-mouse IgG, 1:1000, DAKO) for 1 hour followed. Immunoreactive bands were detected with an enhanced chemiluminescence reagent (ECL Plus, GE Healthcare). Actin (mouse anti-actin AC-40, Sigma) was used as a loading control. The intensity of the resulting protein bands was quantified using MacBiophotonics ImageJ (version 1.47 k). Data is expressed as relative signal intensities (EphA4/Actin) of the individual samples (Figure ). Statistical analyses were performed by t-test analysis for independent samples using GraphPad Prism version 6.0 (San Diego, CA). Furthermore, whole protein extracts were analysed by Immuno-blot analysis with SDS-PAGE using precast stainfree gradient gels (Bio-Rad; Additional file : Figure S6). After UV activation of the gel, proteins were visualised and relative protein amounts were determined and used for normalization. Recombinant proteins EphA1 (75 kDa, aa 569–976; Pro Quinase), EphA4 (72 kDa, aa 586–986; Carna Biosciences) and EphB2 (73 kDa, aa 581–987; Carna Biosciences) were used to proof specificity of the rabbit polyclonal EphA4 antibody. The EphA4 mouse antibody binds extracellularly (aa 379–472) which is outside of the binding domain for recombinant EphA4, therefore only the polyclonal rabbit antibody is shown (Additional file : Figure S8).Figure 1 [...] Pairwise correlation analyses for non-parametric ordinal data were conducted using IBM SPSS Statistics 22.0 (Figure A-D). Spearman’s correlation coefficients and Kendall’s tau coefficients were calculated. Correlations were considered significant in the 95% confidence interval when p < 0.05 (Table ).Figure 5 […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens, Mus musculus
Diseases Alzheimer Disease, Liver Diseases, Neurodegenerative Diseases