Ab40845

The mice were killed respectively at 12 or 42 days after SPS. The brains of mice were quickly resected, and the hippocampal tissues of mice were isolated and immediately maintained at -80°C. The total proteins from mice hippocampus were extracted (Beyotime Biotechnology, China). Then, the protein samples (50 μg/lane) from each group were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Later, protein samples were transferred onto a polyvinylidene fluoride membrane, and were then blocked with 5% defatted milk for 2 h. Afterwards, the membrane was incubated with Glutamate Receptor 1 antibody (1:1000, ab31232), Glutamate Receptor 2 antibody (1:1000, ab206293), Glutamate Receptor 3 antibody (1:1000, ab40845), Glutamate Receptor 4 antibody (1:1000, ab115322), BHLHE40 antibody (1:1000, ab23797), PI3Kp110α antibody (1:1000, ab40776), phosphorylated AKT (ser473) antibody (1:1000, ab8932), AKT antibody (1:1000, ab8805) and GAPDH antibody (1:1000, ab9485) at 4°C overnight, and then incubated with secondary antibodies at room temperature for 2 h. The antibodies were purchased from Abcam (Cambridge, UK). Then, blots were placed to the autoradiography by ECL reagents after washing in Tris buffered saline with Tween. The protein bands were visualized by the Gel Image Analysis System and quantified by ImageJ software (National Institute of Health, USA).