Ndr1 2

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.

The tissue hippocampus was collected from rats at PND30, PND60 and PND90, respectively. The hippocampal protein was obtained by being homogenized in ice-cold lysis buffer (pH7.4; containing 21μg/ml aprotinin, 0.5μg/ml leupetin, 4.9mM MgCl₂, 1mM sodium-Meta-vanandante, 1% Triton X-100 and 1mM PMSF), then centrifugated (14000×g, 7 min), then the supernatant was collected.
The total protein concentration was assayed by the Bicinchoninic Acid (BCA) method. And the equal quality protein of every sample was separated by 8% SDS-PAGE gel before transferring to PVDF membrane. Membranes were blocked with 5% non-fat dry milk, incubated with primary antibodies (GAPDH was purchased from Abcam, ab9484, monoclonal, 1:5000, NDR1/2 was from Santa cruz, sc271703, monoclonal, 1:2000), then washed for 3 times, incubated with the horseradish peroxidase-conjugated secondary antibody and processed for visualization by the enhanced chemiluminescence immuno-blotting detection system. All results were normalized against GAPDH.