Enhanced chemiluminescence system

Protein samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. After blocking with 5% non-fat milk in Tris-buffered saline Tween-20 (TBST), the membranes were incubated with primary antibodies against IL-17 (#AO688, Abclonal Technology, China), Foxp3 (#GB11093, Servicebio, China), phosphoinositide 3-kinase (PI3K) (#bsm-33219m, Bioss, China), phospho (p)-PI3K (Tyr317, #bs-5570R, Bioss, China), protein kinase B (Akt) (#GB11689, Servicebio, China), p-Akt (Ser473, #AF0908, Affinity, USA), FoxO1 (#GB11286, Servicebio, China), p-FoxO1 (Thr24, #9464T, Cell Signaling Technology, USA) or GLP-1R (#CSB-PA009514YA01HU, Cusabio, China) at 4℃ overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies. Proteins were detected using an enhanced chemiluminescence system (Clinx Science Instruments, China).

The hippocampal tissues were homogenized in RIPA buffer containing protease and phosphatase inhibitors, and then centrifuged at 4°C at 12,000 g for 20 min. The concentration of protein in the supernatants was determined using a Bradford protein assay kit (Servicebiotechnology Company, Limited, Wuhan, China). Equal quantities of the protein samples were denatured at 100°C for 15 min, and were then separated by10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred electrophoretically onto a polyvinylidene fluoride membrane (Millipore). The membranes were blocked using 5%-TBST for 60 min and then incubated with the following primary antibodies: mTOR phosphoS2448 (p-mTOR, 1:1,000; Abcam), postsynaptic density protein 95 (PSD-95, 1:1,000; Abcam) and synaptophysin (SYN, 1:1,000; Abcam) at 4°C overnight. Following the membranes were washed three times (5 min each) in TBST and incubated with secondary antibody for 60 min at room temperature. The membranes were detected using an enhanced chemiluminescence system (CLINX, China) and analyzed using AlphaEaseFC (Alpha Innotech).

The 6 paired of tissue samples and cells were homogenized in the lysis buffer containing protease inhibitors and incubated at 4°C for 30 min. After removing precipitated cell debris by high speed centrifugation (12000 rpm for 10 min), the supernatants were added with loading buffer and then boiled at 95°C for 10 min for denature. Approximately 40 μg of each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 10–15% (v/v) polyacrylamide gel. A western blotting system (Bio-RAD) was used to transfer the proteins to polyvinylidene fluoride membrane, which was blocked with 5% (w/v) fat-free milk for 2 h at room temperature and then incubated with an appropriate primary antibody for 12 h at 4°C. The membrane was then washed in Tris-buffered saline containing 0.1% (v/v) Tween^® 20 (TBST) 3 times and incubated with an appropriate secondary antibody conjugated with horseradish peroxidase for 2 h. Finally, the membrane was repeatedly washed in TBST and the bound antibodies were visualized using an enhanced chemiluminescence system (Clinx, Shanghai, China). Immunoblot densitometry and normalization was performed using Image-Pro Plus 6.0 software. Normalized intensity values were compared using the non-parametric Mann-Whitney U test (two-tailed, significance threshold P<0.05) in GraphPad Prism software (version 5.01).