Proteins were extracted as described previously (Liu et al., 2016 (link)). Briefly, hippocampus was homogenized and dissolved in ice-cold lysis buffer (PBS, pH 7.4) containing a cocktail of protein phosphatase and protease inhibitors (21 μg/ml aprotinin, 0.5 μg/ml leupetin, 4.9 mM MgCl2, 1mM sodium-Meta-vanandante, 1% Triton X-100, and 1mM PMSF) to avoid de-phosphorylation and degradation of proteins. Subsequently, all the samples were centrifuged at 14000 ×g at 4°C for 7 min followed by collecting supernatant which was assayed for total protein concentration. Proteins were separated in 8.5% SDS–PAGE gel, and then transferred to PVDF membrane, blocked with 5% non-fat dry milk, followed by incubation with primary antibodies overnight at 4°C. Then membranes were washed for three times, incubated with secondary antibody and then processed for visualization using the enhanced chemiluminescence immuno-blotting detection system. All results were normalized against GAPDH. GAPDH was purchased from Abcam, ab9484, monoclonal, 1:5000, NDR1/2 was from Santa cruz, sc271703, monoclonal, 1:2000.
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