Et1601 4

The hippocampal tissue was placed in the RIPA lysate with PMSF protease inhibitor for homogenization, followed by centrifugation at 12,000 rpm at 4 ℃ to collect the supernatant. A Bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, P0010, Shanghai, China) was used for measuring the protein concentration, and the proteins were subsequently treated with RIPA lysis buffer for further processing. The sample was then boiled for 10 min, cooled and subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) for separation. The protein that has been isolated was moved onto a polyvinylidene fluoride (PVDF) membrane from Merck Millipore (ISEQ00010, USA). After an hour of using 5% skim milk to block the PVDF membrane. And then it was exposed to primary antibodies: SIRT1 (1:1000, 9475S, Cell Signaling Technology), BDNF (1:1000, ab108319, Abcam), and GAPDH (1:1000, ET1601-4, HUABIO) overnight in cold storage at 4 ℃. The membranes were then treated with an antibody conjugate with horseradish peroxidase (1:2000, Beyotime) for an hour at room temperature. The protein bands were then subjected to a quantitative analysis utilizing ImageJ software and an Enhanced Chemiluminescence (ECL) detection system (Beyotime).

Total protein concentration in liver was determined with a BCA Protein Quantification Kit (E112-02, vazyme, Nanjing, China). After boiling denaturation, total protein (35 μg) was separated by 10%~12% SDS-PAGE, then transferred to PVDF membranes (ISEQ00010, Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk for 2 h at room temperature and then incubated (overnight at 4°C) with the following primary antibodies: Rabbit anti-indoleamine 2,3-dioxygenase 1 (IDO-1, 1:1000, 13268-1-AP, Proteintech, Wuhan, China), Rabbit anti-TDO-2 (1:500, 15880-1-AP, Proteintech, Wuhan, China), Rabbit anti-AADAT (KAT2, 1:500, A13090, ABclonal, Wuhan, China) and Rabbit anti-GAPDH (1:8000, ET1601-4, Huabio, Hangzhou, China). Then the HRP conjugated goat anti-rabbit IgG was incubated for 2 h at room temperature. The protein bands were visualized by enhanced chemiluminescence (ECL) reagent (P90720, Millipore, USA) and Gel Image System (Bio-Rad, Hercules, CA, USA). Target signals were analyzed by using Image J software (NIH, Bethesda, MA, USA).

Cells were lysed in RIPA buffer (Solarbio) plus a phosphatase inhibitor cocktail (Epizyme Biotech). The supernatants of lysates were separated by SDS‐PAGE and then blotted on the nitrocellulose blotting membranes (MilliporeSigma, Burkington, MA). The membranes were incubated with primary antibodies against Phospho-STAT3 (1:1000, 9145S, CST), STAT3 (1:1000, 10253-2-AP, Proteintech), MMP2 (1:1000, 10373-2-AP, Proteintech), VEGFA (1:1000, 19003-1-AP, Proteintech), PDGFB (1:1000, AF0240, Affinity) and GAPDH (1:10000, ET1601-4, HUABIO), P16 (1:1000, AF5484, Affinity), P21 (1:1000, 28248-1-AP, Proteintech), γH2A.X (1:1000, ab81299, Abcam), TGFβ (1:1000, sc-130348, Santa Cruz Biotechnology, Inc.), Bax (1:1000, 50599-2-Ig, Proteintech), Bcl2 (1:1000, 3498S, CST), F4/80 (1:500, 14-4801-82, eBioscience), CD31 (1:1000, sc-376764, Santa Cruz Biotechnology, Inc.), OSX (1:1000, A18699, ABclonal) and β actin (1:5000, 20536-1-AP, Proteintech) overnight at 4 °C. Then, the membranes were washed with TBST and incubated with secondary antibodies (1:10000, SA00001-2, Proteintech) for 1 h. The membranes were washed with TBST and then exposed to ECL Chemiluminescent Kit (SQ202, Epizyme Biotech). The signals were quantified using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA).