2.4 Protein levels determination by Western blotting
For protein extraction, tissue samples were homogenised in lysis buffer containing phosphatase and protease inhibitors (Cocktail II, Sigma-Aldrich). Total protein levels were obtained, and protein concentration was determined by the method of Bradford. For WB, aliquots of 15 μg of hippocampal protein were used. Protein samples were separated by Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) (8-16%) and transferred onto Polyvinylidene difluoride (PVDF) membranes (Millipore). Afterwards, membranes were blocked in 5% bovine serum albumin (BSA) in 0,1% Tris-buffered saline - Tween20 (TBS-T) for 1h at room temperature, followed by overnight incubation at 4°C with the primary antibodies listed in Supplementary Table S1. Membranes were washed and incubated with secondary antibodies for 1h at room temperature. Immunoreactive proteins were viewed with a chemiluminescence-based detection kit, following the manufacturer’s protocol (ECL Kit; Millipore) and digital images were acquired using a ChemiDoc XRS+ System (BioRad). Semi-quantitative analyses were carried out using ImageLab software (BioRad), and results were expressed in Arbitrary Units (AU), considering control protein levels as 100%. Protein loading was routinely monitored by immunodetection of glyceraldehyde-3-phosphate dehydrogenase (GADPH) or β-actin.