Synaptophysin

Hypothalamic tissues and cell samples were dissociated in 200 μL of lysis buffer consisting of 1% phenylmethanesulfonyl fluoride (PMSF) (KGP610, KeyGEN, Nanjing, China), and the protein concentration was quantified by a BCA assay kit (Beyotime Biotech Inc., Shanghai, China). The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Roche, Mannheim, Germany). Five percent skim milk in Tris Buffered Saline Tween-20 (TBST) was used to block the membranes for 1 h at room temperature. Following this, the membranes were incubated with the following primary antibodies at 4 °C overnight: PSD95 (1:1000, abcam, Cat# ab12093, Cambridge, UK), Synaptophysin (1:1000, Proteintech, Chicago, IL, USA), FKBP51 (1:1000, Proteintech, Chicago, IL, USA), Lamin B1 (1:1000, Proteintech, Chicago, IL, USA), Progesterone Receptor (1:1000, Bioworld, St. Louis, MO, USA), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000, Proteintech, Chicago, IL, USA). After washing with TBST, the membranes were incubated with appropriate horse radish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. After being washed in TBST 4 times, protein bands were detected by enhanced chemiluminescence and Image J software (Version 1.43, NIH, Bethesda, MD, USA).

Cultured cells were extracted in RIPA buffer (150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1X protease inhibitor cocktail (Roche), phosphatase inhibitor cocktail (Pierce), and 50 mM Tris; pH 8.0). Total protein within the lysate was determined according to the BCA™ protein assay kit (Thermo Scientific, Waltham, MA, USA). The detection and dilution factors of the primary antibodies were as follows: vimentin (1:10,000; Abcam, Cambridge, UK), nicotinic acetylcholine receptor α4 (1:10,000; Abcam), muscarinic acetylcholine receptor 2 (1:1000; Abcam), AMPA receptor (1:250; Abcam), NMDA receptor subunit 2A/NMDAR2A (1:1000; Cell Signaling), dopamine receptor subunit D1 (1:5000; Abcam), synapsin 1 (1:2000; Cell Signaling), synaptophysin (1:10,000; Proteintech, Rosemont, IL, USA), postsynaptic density protein 95/PSD95 (1:1500; BD Bioscience, Heidelberg, Germany), acetylcholinesterase/AChE (1:500; Santa Cruz, Dallas, TX, USA), and actin (1:5000; Sigma-Aldrich). The horseradish peroxidase-conjugated secondary antibodies (Invitrogen) were diluted to a ratio of 1:2000–10,000. Immunolabeled proteins were visualized by using an enhanced chemiluminescence kit (Amersham). Images were taken and analyzed by the UVP ChemStudio PLUS Touch system (Analytik Jena).

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).