Mouse anti glun2b

Protein concentration was determined by Lowry assay. Equal protein concentrations from the fractions were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot for the indicated proteins. Primary antibodies were 1:1000 rabbit anti-GluN2A antibody (Millipore Cat. no. 07-632, RRID:AB_310837), rabbit anti-GluN1 (Millipore Cat. no. AB9864, RRID:AB_11212290), 1:10,000 mouse anti-GluN2B (Cat. no. 610416/7; BD Biosciences), 1:5000 mouse anti-actin (Bio-Rad Cat. no. MCA5775GA, RRID:AB_2571580), mouse anti-Phospho-p44/42 Erk (Tyr204)/(Tyr187) (D1H6G) (Cell Signaling Technology Cat. no. 5726, RRID:AB_2797617), rabbit anti-p44/42 MAPK (Erk1/2) (137F5) (Cell Signaling Technology Cat. no. 4695, RRID:AB_390779). Levels of immunoreactivity were quantified by densitometry using ImageJ 1.49 software (Wayne Rasband, National Institutes of Health, United States).

For western blotting, the procedure was performed as previous study at the age of 6 months [25 (link)]. Analysis of the data was performed using NIH ImageJ software, and the mean density of each band was normalized to β-actin signal in the same sample and averaged. For primary antibodies, we used mouse anti-β-actin (1:3000, Thermo, MA5–15739), rabbit anti-Tau (phosphor-S396) (1:1000, Abcam, ab109390), mouse anti-GluA1 (1:1000, Santa Cruz, sc-13,152), goat anti-GluA2 (1:200, Santa Cruz, sc-7611), mouse anti-GluN1 (1:1000, BD, 556308), rabbit anti-GluN2A (1:1000, Millipore, ab1555P) mouse anti-GluN2B (1:1000, BD, 610417).

For immunoblotting, cortical neurons were harvested in sample buffer comprising 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, and 5% β-mercaptoethanol and heated for 5 min at 95 °C. Proteins were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes at 80 V for 1.5 h. The membranes were incubated with 5% nonfat milk in 10 mM Tris-HCl, pH 7.4, containing 0.9% NaCl and 0.1% Tween 20 for 1 h at room temperature, and then incubated overnight at 4 °C with primary antibodies. Subsequently, the membranes were probed with horseradish peroxidase-conjugated secondary antibodies (dilution, 1:5000; Pierce Biotechnology, Rockford, IL, USA). Immunoreactive proteins were detected by use of ImmunoStar basic (Wako), ImmunoStar zeta (Wako) or West Femto (Pierce Biotechnology). The following primary antibodies were used: mouse anti-β-actin (a5441, Sigma), mouse anti-GluN1 (556308, BD Biosciences, Franklin Lakes, NJ, USA), rabbit anti-GluN2A (AB1555P, Millipore), mouse anti-GluN2B (610416, BD Biosciences), and rabbit anti-calpain-2 (39165, Abcam).