Western blots were performed as described previously [43]. Briefly, the dissected mPFC samples were homogenized in a lysis buffer (Beyotime Biotechnology, Shanghai, China) with 1% phenylmethylsulfonyl fluoride (Sangon Biotech, Shanghai, China) and 1 × PhosSTOP phosphatase inhibitor cocktail (Roche Applied Science, Penzberg, Germany). The extracted proteins were separated on a 15% SDS-PAGE gel and transferred to PVDF membrane (Millipore, Massachusetts, USA). The membrane was blocked with 5% non-fat milk powder in TBST buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween20), followed by incubation with the following primary antibodies diluted in a blocking solution overnight at 4 °C: anti-BDNF (sc-546, 1:500, Santa Cruz Biotechnology, Dallas, TX, USA), anti-FoxO1 (1:500, #2880, Cell Signaling Technology, Danvers, MA, USA), or anti-β-actin antibody (1:1000, #4970, Cell Signaling Technology, Danvers, MA, USA). After washing, the membrane was incubated with IRDye 680LT donkey anti-rabbit IgG secondary antibodies (1:5000; 926–68,023, Li-COR Biosciences, Lincoln, NE, USA). The fluorescence was visualized and analyzed using an Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE, USA).
