Western Blot Analysis of Synaptic Proteins

Synaptosomes or the frozen hippocampus tissues were suspended in ice-cold lysis buffer and quantified for protein content [24 (link)]. Thirty micrograms of protein was subjected to 8–12% SDS-PAGE, and proteins were electroblotted to nitrocellulose membranes. After blocking with 5% nonfat milk for 1 h, membranes were incubated overnight at 4 °C with the following antibodies: anti-phospho-ERK1/2 (p-ERK1/2,1:2000, Cell Signaling), anti-ERK1/2 (1:4000, Cell Signaling), anti-HSP70 (1:2000, Enzo), anti-Akt (1:6000, Cell Signaling), anti-phospho-Akt (p-Akt,1:6000, Cell Signaling), and anti-β-actin (1:8000, Cell Signaling). Membranes were then washed three times with Tris-buffered saline and incubated for 1 h at room temperature with suitable secondary antibodies. After washing, the blots were developed using an enhanced chemiluminescence detection system (Amersham, Berkshire, UK), which was followed by analysis using the Image J software (version 1.45 J, National Institutes of Health, Bethesda, Rockville, MD, USA).