Western blot analysis of BDNF, and its precursor proBDNF, TrkB, and phosphorylated-TrkB

Western blot analysis was performed as reported previously (Ren et al. 2015, 2016; Yang et al. 2015b; Zhang et al. 2015a, 2015b). Mice were killed by cervical dislocation and brains were rapidly removed from the skull. Approximately 1 mm thick coronal sections were cut and bilateral tissue punches of prefrontal cortex (PFC), nucleus accumbens (NAc), striatum, CA1, CA3, and dentate gyrus (DG) of the hippocampus were dissected on ice using a SZ-LED Kenis light microscope (Osaka, Japan), and stored at −80 °C. Tissue samples were homogenized in Laemmli lysis buffer. Aliquots (20 μg) of protein were measured using the DC protein assay kit (Bio-Rad), and incubated for 5 min at 95 °C, with an equal volume of 125 mM Tris-HCl, pH 6.8, 20% glycerol, 0.1% bromophenol blue, 10% β-mercaptoethanol, 4% SDS, and subjected to SDS polyacrylamide gel electrophoresis using AnyKD minigels (Mini-PROTEAN TGX Precast Gel; BioRad). Proteins were transferred onto PVDF membranes using a Trans Blot Mini Cell (Bio-Rad). For immunodetection, the blots were blocked with 2% BSA in TBST (TBS + 0.1% Tween-20) for 1 h at room temperature, and kept with primary antibodies overnight at 4 °C. The following primary antibodies were used: BDNF (1:200; H-117, Cat#: sc-20981, Santa Cruz Biotechnology), phosphor-TrkB (Tyr-706) (1:200; Cat#: sc135645, Santa Cruz Biotechnology), TrkB (80E3) (1:1000; Cat#: 4603, Cell Signaling Technology). The next day, blots were washed three times in TBST and incubated with horseradish peroxidase-conjugated anti-rabbit antibody (1:10,000) 1 h at room temperature. After a final three washes with TBST, bands were detected using enhanced chemiluminescence (ECL) plus the Western Blotting Detection system (GE Healthcare Bioscience). The blots were then washed three times in TBST and incubated with the primary antibody directed against β-actin (1:10,000; Sigma-Aldrich). Images were captured with a Fuji LAS3000-mini imaging system (Fujifilm, Tokyo, Japan), and immunoreactive bands were quantified.