Cortex (A) and hippocampus (B) proteins extracts obtained from wild type (WT) and triple transgenic AD mice model (3xTg-AD) at 6 and 12 months of age were subjected to immunoblot analysis with the indicated phospho and total antibodies

Cortex (A) and hippocampus (B) proteins extracts obtained from wild type (WT) and triple transgenic AD mice model (3xTg-AD) at 6 and 12 months of age were subjected to immunoblot analysis with the indicated phospho and total antibodies. singularly also contribute to protect these mice against amyloid-beta pathology. access to food and water. All animal procedures were performed in accordance with 2010/63/UE regarding the care and use of animals for experimental APG-115 procedures following the recommendations of the Universitat Autnoma de Barcelona Ethical Committee for Human and Animal Experimentation. The protocols were approved by the Ethical Committee APG-115 for Human and Animal Experimentation, Universitat Autnoma de Barcelona, and performed under a Generalitat de Catalunya Project License. The study complies with the ARRIVE guidelines developed by the NC3Rs (Kilkenny et al., 2010). APG-115 Primary cultures of embryonic neurons and cell assays Neuronal primary cultures were established from embryonic day 15,5 (E15,5) embryos as previously described (Zurashvili et al., 2013). Cortical cells were seeded at a density of 5 104 cells/cm2 onto 50 g/ml of poly-D-Lysine-coated 24-well plates for cell viability studies, or 6-well plates for western blot analysis, and grown for 6 days in a 5% CO2 incubator. The hippocampal cells were plated at a density of 2 104 cells/cm2 on 24-well plates made up of 12-mm glass coverslips coated with 150 g/ml of poly-D-Lysine for 4 days in a 5% CO2 incubator. Stock solutions of tunicamycin and Akti-1/2 dissolved in dimethyl sulfoxide, or TNF and A dissolved in PBS, were added to cultures as indicated. Cell survival was determined by the MTT reduction assay as described previously (Zurashvili et al., 2013). For the quantification of apoptosis, cells were fixed in 2% paraformaldehyde, stained with 1 g/ml of the DNA dye Hoechst 33342, and then visualized under the fluorescence microscope. Apoptosis was quantified at each condition point by scoring the percentage of cells exhibiting fragmented or condensed nuclei. At least 300 cells per well from 6 systematically collected fields starting from a APG-115 random position were manually counted by two participants, with blinding to the treatment conditions. Generation of protein extracts and western blot analysis Tissue extracts were prepared by homogenizing the frozen tissue in a 10-fold excess volume of ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 10 mM sodium-glycerophosphate, 0.27 M sucrose, 1% w/v Triton X-100, 0.1% v/v 2-mercaptoethanol, and a 1:100 dilution of protease inhibitor cocktail, (Sigma, Merck KGaA, Darmstadt, Germany) using the Polytron homogenizer; cultured cortical neurons were scrapped from wells in the same ice-cold lysis buffer. Lysates were centrifuged at 4C for 15 min at 15,000 g and supernatants aliquoted and preserved at ?20C. Protein concentrations were determined by the Bradford method using bovine serum albumin as standard. The activation state of the different pathways was assessed by loading 20 g of protein extracts onto 6C10% polyacrylamide, 0.25M Tris-HCl pH 8.5, 0.1% (w/v) SDS gels casted in 10-well/1 mm-thick mini-PAGE (polyacrylamide gel electrophoresis) system (Bio-Rad, Hercules, California, USA), then electrophoresed on 25 mM Tris-HCl pH 8.5, 192 mM Glycine, 0.1% (w/v) SDS running buffer at 160 V for CD253 60 min, and finally transferred to nitrocellulose membranes (Whatman, Maidstone, UK) on a 25 mM Tris-HCl pH 8.5, 192 mM Glycine, 20% methanol transfer buffer wet system at 100 V for 90 min. Samples were blocked with 10% skimmed milk, 1xTBS, 0.2% (w/v) Tween 20 for 1 h at room temperature, then immunoblotted over night at 4C with 1 g/ml of the indicated primary antibodies diluted in either 5% skimmed milk, 1xTBS, 0.2% (w/v) Tween 20 (total protein antibodies) or 0.5% BSA, 1xTBS, 0.2% (w/v) Tween 20 (phospho-antibodies), which were detected with a 1:5,000 dilution of the appropriate horseradish peroxidase-conjugated secondary antibodies diluted in 5% skimmed milk, 1xTBS, 0.2% (w/v) Tween 20. Membranes were incubated with the enhanced chemiluminescence (ECL) reagent, then either exposed to Super RX Fujifilm and developed, or detected using a ChemiDoc MP Imaging System (Bio-Rad, Hercules, California, USA), and quantified using the ImageJ software. Membrane and cytosol preparation Cortex tissues were APG-115 homogenized in 10 volumes of cold homogenization buffer (50 mM Tris-HCl pH 7.4, 0.27 M sucrose, 1 mM Na3VO4, 1 mM EDTA, 1 mM EGTA, 50 mM.