Fractionation and Western Blot Analysis of Mouse Brain Regions

Cortex, prefrontal cortex, hippocampus, cerebellum, striatum and hippocampal PSD fractions were prepared from three pairs of 8-week-old Ctrl and cKO male mouse brains as previously described61 (link). Adult neural progenitors were isolated from adult mouse hippocampi (C57BL/6) and cultured as previously described62 (link)63 (link). Protein lysate subjected to western blot analysis were separated on SDS–polyacrylamide gel electrophoresis and probed with specific antibodies; GluN1 (rabbit, 1:1,000; Epitomics, 2824-1,), GluN2A (Cell Signaling Technology, 4205, 1:1,000), GluN2B (Cell Signaling Technology, 4212, 1:1,000), GluA1 (rabbit, 1:1,000; Epitomics, 3861-1), GluA2 (rabbit, 1:1,000; Epitomics, 3520-1), CRMP2 (rabbit, 1:1,000; Cell Signaling Technology, 9393 and Sigma,), p-CRMP2 (rabbit, 1:1,000; Epitomics, 5799-1), SYP (mouse, 1:1,000; Santa Cruz, Sc-17750), PSD-95 (rabbit, 1:1,000; Abcam, ab18258), Actin (mouse, 1:1,000; Cell Signaling Technology, 3700), GAPDH (rabbit, 1:1,000; Cell Signaling Technology, 2118). The relative amount of β-Actin or GAPDH was used as loading control. For quantification, the densitometry measurement of each band (Image J) was first normalized to that of Actin or GAPDH and then averaged from at least three independent experiments. Images have been cropped for presentation. Full size images are presented in Supplementary Fig. 4.