Glua2

Mice from each group were decapitated at ZT3, and their brains were collected. Hippocampal tissues were isolated; weighed; successively treated with lysis buffer (10 mL/g), a broad spectrum phosphatase inhibitor (1 : 100) and PMSF (1 : 100); and ultrasonicated on ice. The supernatant was stored at 4°C for 4 h and centrifuged at 13000 r/min for 15 min, and the protein concentration was determined by the BCA assay. Thirty micrograms of protein was heated for 5 min, separated by SDS-PAGE, and transferred onto a polyvinylidene fluoride (PVDF) membrane, which was incubated with an anti-rabbit anti-polyclonal antibody (1 : 500) or mouse anti-β-actin antibody (1 : 500) overnight at 4°C. Then, the membrane was incubated with an HRP-labeled goat anti-rabbit IgG (1 : 5000) or HRP-labeled goat anti-mouse IgG (1 : 5000) secondary antibody at 4°C for 2 h and ECL reagent was added for development. ImageJ software was used to analyze the levels of OxA, cAMP, PKA, CREB, phosphorylated CREB (pCREB), GluN1, GluN2A, GluA2, synaptophysin (SYP), and postsynaptic density protein-95 (PSD95) (all antibodies are purchased from Cell Signaling Technology, Danvers, MA).

Antibodies used in this study included the polyclonal rabbit antibodies against NR2B (#4207, Cell Signaling Technology, Beverly, MA, USA), NR1 (#5704, Cell Signaling Technology), GluA1 (#13185, Cell Signaling Technology), GluA2 (#13607, Cell Signaling Technology), GluA3 (#3437, Cell Signaling Technology), GluA4 (#8070, Cell Signaling Technology), PSD-95 (#3450, Cell Signaling Technology), phospho-Cofilin (#3311, Cell Signaling Technology), Cofilin (#5175, Cell Signaling Technology), Arp3 (#4738, Cell Signaling Technology), NR2A (#05-901R, Millipore, Merck KGaA, Darmstadt, Germany), NR2C (#OPA1-04020, Millipore), NR2D (#PA5-87624, Millipore), AKAP150 (#07-210, Millipore), HMGB1 (#10829-1-AP, Proteintech, Rosemont, IL, USA), Calnexin (#10427-2-AP, Proteintech), Synaptophysin (#17785-1-AP, Proteintech), Iba-1 (#019-19741, Wako Chemicals, Osaka, Japan), and the monoclonal mouse antibodies against NR2A (#MA5-27692, Invitrogen, Carlsbad, CA, USA), NR2B (#MA1-2014, Invitrogen), β-actin (#66009-1-Ig, Proteintech), GAPDH (#60004-1-Ig, Proteintech), Syntaxin (#66437-1-Ig, Proteintech), NeuN (#MAB377, Millipore), RhoA (#ARH04, Cytoskeleton Inc., Denver, CO, USA), Rac1 (#ARC03, Cytoskeleton), Cdc42 (#ACD03, Cytoskeleton), and Rac1-GTP (#26903, Neweast Biosciences, Wuhan, China). Pharmacological agents included glycyrrhizin (#50531, Sigma, St. Louis, MO, USA).