T1082

Total protein was extracted from the hippocampus in RIPA lysis (C1053, APPLYGEN, China) buffer containing a protease inhibitor and supplemented with phosphatase inhibitors (11836170001, Roche, Germany), and centrifuged at 12,000 × g for 5 min at 4°C. The protein was quantified by the BCA protein assay kit. Protein lysates were cleared of insoluble material through centrifugation. Proteins were wet transferred to 0.2 μm pore size, hydrophobic PVDF transfer membranes (ISEQ00010, Millipore, Germany), which were blocked using 5% non-fat milk in 1% TBST buffer for 1 h at room temperature. The membranes were incubated overnight using the following primary antibodies: O-GlcNAc (CTD110.6, 1:1000 dilution) Mouse mAb (12938S, CST, USA), GAPDH (1:5000 dilution) Mouse McAb (60004-1, Proteintech, China). All primary antibodies were used in 5% BSA (CZ1006, czkwbio, China). Membranes were washed in 1%TBST (T1082, Solarbio, China) and incubated with the following appropriate secondary antibodies: goat anti-mouse HRP. The secondary antibodies were used at a 1:5,000 and 1:10000 dilution in 5% BSA. Protein bands were visualized following exposure of the membranes to ECL (RM0021, ABclonal, China) and quantified by densitometry analysis using Image software.

PGCs were collected and lysed using Radio-Immunoprecipitation Assay (RIPA) lysis solution (Beyotime-P0013B, Shanghai, China). Extracted total proteins were separated and transferred to PVDF membrane (Beyotime-FFP39, Shanghai, China) via electrophoresis. Blots were blocked using 5% skimmed milk at room temperature for 2 h, followed by the incubation of primary antibody solution (PI3K (BIOSS-bsm-33219M, Beijing, China), p-PI3K (BIOSS-bs-6417R, Beijing, China), AKT (Affinity-AF6261, Liyang, China), p-AKT (BIOSS-bs-0876R, Beijing, China), GAPDH (Proteintech-10494-1-AP, Wuhan, China)) at 4 °C overnight. After 3 washes with TBST, the blots were incubated with secondary antibody solution (Goat Anti-Mouse IgG (CWBIO-CW0102, Taizhou, China), Goat Anti-Rabbit IgG (CWBIO-CW0103, Taizhou, China) at room temperature for 2 h. The blots were washed 3 times with TBST (Solarbio-T1082, Beijing, China) and subjected to imaging using chemiluminescent substrate.