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.