Glun2b

Mice were sacrificed on the last treatment day with rTMS. The peri‐infract cortex tissues (approximately 20 mg) were homogenized in 200 μL of RIPA lysis buffer (P0013B, Beyotime, Shanghai, China). Protein quantification was performed using a BCA protein assay kit (Melone Pharmaceutical, Dalian, China). Samples with 40 μg protein were separated by size using 10% sodium dodecyl sulfate–polyacrylamide gels and transferred onto nitrocellulose (NC) membranes. Membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: integrin α3 (1:1000, 66,070–1, Proteintech), GluN2B (1:1000, 21,920‐1‐AP, Proteintech), β‐actin (1:1000, SD0034, SIMUWU), GluN2A (1:1000, 19,953‐1‐AP, Proteintech), GluA2/3/4 (1:1000, 2460, Cell Signaling), GluA1 (1:1000, 67,642‐1‐Ig, Proteintech), GAD65 (1:1000, 20,746‐1‐AP, Proteintech), and vGlut1 (1:1000, ab227805, Abcam). Membranes were washed with TBST and incubated with HRP‐conjugated goat anti‐mouse and goat anti‐rabbit antibody for 1 h at room temperature. ImageJ software was used to analyze protein bands.

Antibodies against RAB39B (Cat# D-12162-1-AP), GluA2 (Cat# 11994-1-AP), GluN2A (Cat# 19953-1-AP), GluN2B (Cat# 19954-1-AP), and tyrosine hydroxylase (TH, Cat# 66334-1-IG) were from Proteintech. Antibodies against GFAP (Cat# 3670s), β-actin (Cat# 8457s), p62 (Cat# 5114s), GluN1 (Cat# 5704s), PSD95 (Cat# 3450s), LC3B (Cat# 3868s), S6 (Cat# 2217s), and phosphorylated S6 (Ser240/244) (Cat# 5364s) were from Cell Signaling Technology. Antibodies against GluA1 (Cat# 04-855), GluA3 (Cat# MAB5416), and SYN1 (Cat# AB1543) were from Millipore. Antibodies against NeuN (Cat# ab177487), Iba1 (Cat# 016-20001), and synaptophysin (SYP) (Cat# S5768) were from Abcam, Wako, and Sigma-Aldrich, respectively. Goat anti-rabbit (Cat# 31460) or anti-mouse (Cat# 31430) IgG (HCL) secondary antibodies conjugated with horseradish peroxidase were from Thermo Fisher Scientific.
The mTOR inhibitor rapamycin (Cat# HY-10219) was from MedChemExpress. DMSO (Cat# 20688) was from Thermo Fisher Scientific. 4′,6-diamidino-2-phenylindole (DAPI) (Cat# D9542) was from Sigma-Aldrich. Complete Protease Inhibitor and PhosSTOP Cocktails were from Roche.
Different protein levels in mouse tissues and cells were determined by western blot. Detailed procedures are presented in Supplementary Materials and Methods. Protein band intensity was quantified by densitometry using the Image J software (National Institutes of Health).