Quantifying Protein Levels via Western Blot

To quantify protein levels, western blotting was carried out as previously described (Autry et al., 2011 (link); Gideons et al., 2014 (link); Suzuki et al., 2017 (link)). To measure protein level in cultured neurons, cultured hippocampal neurons were lysed in Laemmli sample buffer containing phosphatase and protease inhibitors (Roche). To quantify protein level in hippocampus, hippocampus was dissected and lysed using RIPA buffer [50 mM Tris, pH 7.4, 1% Igepal® CA-630, 0.1% SDS, 0.5% sodium deoxycholate, 4 mM EDTA, 150 mM NaCl, phosphatase and protease inhibitors (Roche)], and lysate was mixed with Laemmli sample buffer. Samples were boiled for 5 min at 95°C. Then samples were loaded onto SDS-PAGE gels and transferred to nitrocellulose membranes. Primary antibodies were incubated with membrane at following dilutions: 1:500 for anti-eEF2K (Abcam, ab45168), 1:5,000 for anti-phosphorylated eEF2 (Thr56) (Cell Signaling, 2331), 1:2,000 for anti-total eEF2 (Cell Signaling, 2332), 1:1,500 for anti-BDNF (Abcam, ab108319), 1:20,000 for anti-ERK1/2 (Cell Signaling, 4695), 1:20,000 for anti-phospho-ERK1/2 (Cell Signaling, 4370) and 1:50,000 for anti-GAPDH (Cell Signaling, 2118). Membranes were incubated with HRP anti-rabbit secondary antibody (Vector Laboratories, Inc.). Chemiluminescence was detected using Clarity Western ECL substrate (Bio-Rad). Band intensities were analyzed by ImageJ