Las 2000 system

Synaptosome purification has been described previously^{26 (link)}. For the validation of NBEA expression after retrieval, whole hippocampal tissue was used. For the rest of the study, the dorsal hippocampus was dissected and homogenized in ice-cold lysis buffer using a Teflon-glass tube. For immunoblotting analysis, antibodies targeting the following proteins were used. Primary antibodies against the following antigens were used: synapsin I (1:1,000, Abcam, Cambridge, UK), postsynaptic density 95 (PSD95; 1:1,000, Abcam, Cambridge, UK), histone H1 (1:1,000, Abcam, Cambridge, UK), NBEA (K-20) (1:1,000, Santa Cruz Biotech., Dallas, TX, USA), A kinase anchor protein 150 (AKAP150; 1:1,000, Santa Cruz Biotech, Dallas, TX, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5,000, Cell signaling, Danvers, MA, USA). The secondary antibodies were horseradish peroxidase (HRP)-conjugated anti-mouse (1:5,000) or anti-rabbit (1:10,000) antibodies (PerkinElmer Life Sciences, Norwalk, CT, USA). Signal detection was performed using the LAS 2000 system (LAS-2000, Fuji, Tokyo, Japan) with enhanced chemiluminescence (Western-CDP star, PerkinElmer Life Sciences, Norwalk, CT, USA). Densitometric analysis of immunoreactivity for each protein was conducted using NIH ImageJ software.

Western blot analysis was performed as previously described [1 (link)] using BDNF (1:2,000; GeneTex, Irvine, CA, USA) and GAPDH (1:5000; Cell Signaling, Danvers, MA, USA) antibodies. Signal detection was performed using the LAS-2000 system (LAS-2000; Fuji, Tokyo, Japan) with enhanced chemiluminescence (Western-CDP star; PerkinElmer Life Sciences, Norwalk, CT, USA). Densitometric analysis of immunoreactivity for each protein was conducted using NIH ImageJ software.