Hippocampal Protein Expression Analysis by Western Blot

Western blotting was performed as previously described [36 (link)]. Briefly, mouse hippocampal samples were homogenized in ice-cold IGEPAL RIPA buffer with protease inhibitors at 4 °C. 20 or 40 µg of total protein in Laemmli loading buffer was denatured at 85 °C for 10 min and separated by 7.5-, 10-, or 12% Criterion TGX Stain-free SDS-PAGE (Bio-Rad) and blotted onto 0.45 µm nitrocellulose membrane (Bio-Rad) by submerged transfer apparatus (Bio-Rad). Membranes were then blocked and incubated overnight with gentle agitation at 4 °C with the primary antibody (Additional File 1: Table S1). Protein was detected by chemiluminescence (Clarity, Bio-Rad) according to the manufacturer’s instructions and signals were captured on a ChemiDoc Touch MP Imaging System (Bio-Rad).