E021040 01

Tissue blocks of mPFC and NAc shell were punched 4 h and 1 h after intra-VTA injection respectively. The tissues were homogenized in lysis buffer containing protease and phosphatase inhibitors, followed by centrifugation (13000 rpm, 15 min, 4°C). With the supernatants collected and protein concentrations measured, the supernatants were adjusted to the identical protein concentration across groups. The samples (40 μg protein per lane) were electrophoresed by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). The membranes were thrice rinsed and blocked in 5% w/v skim milk for 2h at 25°C, followed by incubation in primary polyclonal rabbit anti-BDNF (1:1000, sc-546, Santa Cruz, USA,) or polyclonal rabbit anti-β-Tubulin (1:1000, E021040–01, Earthox, USA) antibody overnight at 4°C. Afterwards, the bands were rinsed and incubated in alkaline phosphatase-labeled secondary antibody (1:1000, AP-1000, VECTOR, USA) (2h, 25°C), and were visualized by a BCIP/NBT kit (S3771, Promega, USA). We detected a band of approximately 15 kDa, indicating truncated BDNF. β-Tubulin served as the standard of comparison and the gray scale intensity of bands was analyzed by ImageJ software.