Imagelab 1

Total mPFC tissue was collected 1 hour after the last injection, and the protein concentration in each sample was determined using a BCA protein assay kit (Pierce, Rockland, IL, USA). These protein samples were separated using 10% SDS–PAGE and transferred to nitrocellulose membranes (Millipore, USA). Then, the membranes were blocked for 3 h in 5% (w/v) milk at room temperature and incubated overnight at 4 °C with the following primary antibodies: rabbit anti-Notch1 (1:1000, Abcam, ab8925), rabbit anti-Hes1 (1:1000, Abcam, ab71559), and mouse anti-GABA_B1 receptor (1:2000, Abcam, ab55051). Membranes were then washed with TBST and probed with the appropriate horseradish peroxidase-conjugated secondary antibodies (1:2000) for 1.5 h at room temperature. Proteins were detected with an enhanced chemiluminescence assay kit (ECL Plus, Millipore Corporation, USA). Signals were visualized using ImageLab 1.46 (BioRad, USA).

Protein homogenates were prepared with 5× protein loading buffer (HEART R0891, Xi’an, China) and denatured at 95°C for 5 min. Fifteen micrograms of protein per sample was resolved on a precast 10% (w/v) SDS-PAGE gel and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore IPVH00010, Bedford, MA, United States). Blots were blocked with 5% (w/v) nonfat milk solution (in Tris-buffered saline with 0.1% Tween-20 (TBST)) and then incubated overnight at 4°C in primary antibody solutions (Anti-Ago2, Abcam, ab186733, diluted 1:2000). Membranes were then washed with TBST and probed with the appropriate horseradish peroxidase-conjugated secondary antibodies (1:2000) for 1 h at room temperature. Membranes were visualized using an enhanced chemiluminescence detection kit (Solarbio PE0010, Beijing, China) and quantified with ImageLab 1.46 (BioRad, United States).