2.12 Western blotting experiments
The experiment was processed according to previous reports with some modifications(Taylor & Posch, 2014). Animals were killed the day after behavioral studies. To extract the total proteins, bilateral NAc were immediately dissected and homogenized in 10 mM Tris HCl pH 8, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS containing Complete Mini protease inhibitor cocktail (Snyder & Kim), and then kept on ice for 30 min(Ntoukas et al., 2020). The lysates were centrifuged and supernatants were collected. Then, the samples were mixed with loading buffer and boiled for 5 min. Protein concentration was estimated using the BCA Protein Assay (Pierce). After denaturation, equal amounts of protein samples (30 mg) were separated by 10% SDS/PAGE gel and then transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). After being blocked with 10% non-fat dried milk powder/Tris-buffered saline Tween-20 (TBST) for 1 h, membranes were incubated overnight at 4℃with primary antibodies to BDNF (1:500; Abcam, UK), TrkB (1:1000; Abcam, UK), phospho-TrkB-tyr515 (p-TrkB;1:500; Abcam, UK), cAMP response element-binding protein CREB (1:1000; Abcam, UK), phospho-CREB-Ser133 (pCREB; 1:500; Abcam, UK), and β-actin (1:1000; Abcam, UK). Then primary antibodies were removed by washing the membranes three times in TBST. The membranes were further incubated for 2 hours at room temperature with IRDye 680-labelled secondary antibodies (1:2000; Santa Cruz). Finally immunoblots were visualized by using enhanced chemiluminescence (ECL; Pierce, Rockford, IL, USA). The optical density of the bands was quantified using ImageJ software (Packard Instruments BV, Groningen, Netherlands). The results were normalized to the quantity of β -actin in each sample lane. All assays were performed at least three times.