Ab259880

Immediately after the rats were euthanized (Figure 1B), their mPFC, CeA, and hippocampal CA1 were retrieved immediately through dissection and stored at –80°C until analysis (Li et al., 2016 (link), 2017 (link)). The tissues were homogenized and lysed using a radio-immuno-precipitation assay buffer (AR0102, Boster) and then subjected to the measurement of protein concentration using the bicinchoninic acid kit (AR1189, Boster). The lysates (20 μg each) were separated on 10% SDS-PAGE gels, and the separated protein bands were transferred electrophoretically to a polyvinylidene fluoride (PVDF) membrane. Immunoreactivity was determined based on enhanced chemiluminescence, and the signals were detected using a Bio-Rad ChemiDoc system (Bio-Rad Laboratories, China).
The following antibodies were used: anti-GAPDH (1:5,000, BM1623, Boster); anti-GluA1 (1:1,000, A1826, ABclonal); anti-mGluR1 (1:1,000, abx112750, Abbexa); anti-mGluR5 (1:1,000, A3758, ABclonal); anti-GluN2B (1:1,000, abx23583, Abbexa); anti-PSD-95 (1:1,000, abx236850, Abbexa); anti-α5 GABA (1:1,000, ab259880, Abcam). The secondary antibodies used were goat anti-rabbit IgG (1:10,000; Boster) and goat anti-mouse IgG (1: 10,000, Boster). The immunosignals were quantified using densitometry and were expressed relative to the GAPDH signals and normalized to the control for data analysis.

A radioimmunoprecipitation assay buffer (RIPA; Solarbio, R0010) supplemented with phenylmethanesulfonyl fluoride (PMSF; Solarbio, P0100) was used to lyse the cultured neuronal cells and hippocampal tissue in the rat brain. In a subset of experiments, Mem‐PER Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific) was used to extract membrane proteins. The protein concentration was determined using bicinchoninic acid (BCA) protein detection kit (Beyotime Biotechnology). After denaturation, equal amount of protein samples was separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, 0.45 μm). After blocking with 5% skimmed milk, membranes were sequentially incubated with primary antibodies [BDNF (1:1000, Cell Signaling Technology, #47808), GABA_AR α1 (1:1000, Abcam, #ab94585), GABA_AR α5 (1:1000; Abcam, #ab259880), and GAPDH (1:1000; Abcam, #ab8245)] and secondary antibodies (goat anti‐rabbit IgG H&L, HRP; 1:2000; Abcam, #ab6702). Bands were visualized with an enhanced chemiluminescence detection kit (EMD Millipore). Using Image‐Pro Plus (Media Cybernetics), the band density was analyzed and standardized as fold of GAPDH.