Bicinchoninic acid kit

Following brain removal after deep anesthetization with sodium pentobarbital, the mPFC, hippocampus, and CeA were homogenized in lysis buffer. After centrifugation, the protein concentration of the supernatant was detected by using the bicinchoninic acid kit (Boster). Then, 20 μg of each nuclear protein sample was separated on 10% SDS-PAGE gels and transferred onto a polyvinylidene fluoride membrane, which was then blocked in 5% skim milk for 2 h at room temperature, and the membrane was incubated overnight at 4 °C with different primary antibodies. Then, the blots were washed thrice and incubated with the secondary antibody for 1 h at room temperature. Immunoreactivity was quantified by densitometry with enhanced chemiluminescence.
The following primary antibodies were used: anti-GAPDH (1:1000, A19056, ABclonal); anti-ACSS2 (1:1000, T55995, Abmart); anti-HDAC2 (1:1000, 57156S, CST); anti-Ace-H4K12 (1:1000, 13,944, CST); anti-Ace-Lysine (1:1000, 9441, CST).

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.

PC12 cells were harvested and lysed using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Nantong, China). Total proteins were extracted and quantified using a bicinchoninic acid kit (Boster Biological Technology, Wuhan, China). Subsequently, 50 μg proteins were fractionated using 10% SDS-PAGE (GE Healthcare, Logan, UT, USA) and electro-transferred onto nitrocellulose (NC) membranes (Bioleaf Biotech, Shnghai, China; in an ice-water bath. NC membranes were blocked with 5% skimmed milk in Tris buffer (Sigma-Aldrich) containing 0.1% Tween-20 and then incubated with the following rabbit monoclonal primary antibodies at 4°C overnight: Anti-Bax (sc-526), anti-Bcl-2 (sc-492), anti-extracellular-signal-regulated kinase (ERK; sc-292838), anti-phosphorylated (p)-ERK (sc-13073), anti-caspase-3 (all Santa Cruz Biotechnology Inc.), and anti-p53 (9282S), anti-Akt (9272) and anti-p-Akt (9275) (Cell Signaling Technology, Inc.) (all 1:1,000 dilution). Subsequently, the blots were washed and incubated with goat anti-rabbit horseradish peroxidase-conjugated immunoglobulin G secondary antibody (Santa Cruz Biotechnology) at room temperature for 1 h. Finally, blots were visualized with enhanced chemiluminescence reagent (EMD Millipore).

Cell protein was extracted, and protein concentration was determined as per the instructions of the bicinchoninic acid kit (Boster Biological Technology Co., Ltd., Wuhan, Hubei, China). Following the addition of loading buffer (30 μg/well), the extracted protein underwent 10 min boiling at 95°C. The protein was then subjected to 10% polyacrylamide gel (Boster) electrophoresis (voltage changed from 80 V to 120 V) for separation and then loaded onto polyvinylidene fluoride membranes via wet transfer at a voltage of 100 mV for 45–70 min. Following 1 h blocking with 5% BSA at room temperature, the membranes underwent overnight incubation at 4°C with primary antibodies phosphatase and tensin homolog (PTEN) (1 : 1000, ab267787, Abcam), light chain 3 (LC3)I, LC3II (ZSGB-Bio Co., Ltd, Beijing, China), p62 (2 μg/mL, ab56416, Abcam), Beclin-1 (1 : 2000, ab207612, Abcam), and GADPH (1 : 2500, ab9485, Abcam). Following 3 washes with Tris-buffered saline-Tween 20 (5 min per wash), the membranes underwent 1 h incubation with a secondary antibody IgG (1 : 2000, ab205718, Abcam) at room temperature. The membranes were washed (3 times, 5 min each) and developed using chemiluminescence reagent and GelDOC EZ Imager (Bio-Rad Inc., Hercules, CA, USA). ImageJ software (National Institutes of Health, Bethesda, Maryland, USA) was utilized to analyze the gray value of the target bands.

The cultured cells were collected and lysed with enhanced radioimmunoprecipitation assay lysis buffer (Boster Biological Technology, Wuhan, P.R. China) containing protease inhibitors. The bicinchoninic acid kit (Boster Biological Technology) was utilized to determine the protein concentration. Proteins were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the separated proteins were electro-transferred to polyvinylidene fluoride membrane (Immobilon P, Millipore, Billerica, MA, USA), and the membrane was treated with 5% skimmed milk at ambient temperature for 2 h to block non-specific binding. The membrane was incubated with primary antibody at 4°C overnight and then with horseradish peroxidase conjugated secondary antibody at 37°C for 1 h. Enhanced chemiluminescence reagent (Thermo Fisher Scientific) was utilized to visualize the immunoreactive bands, followed by imaging using ChemiDoc XRS Plus luminescent image analyzer (Bio-Rad, Hercules, CA, USA). ImageJ analysis software was utilized to quantify the gray value of protein bands. Antibodies included SIRT6 (A7416, 1:2,000, ABclonal, Woburn, MA, USA), NRP-1 (A19087, 1:2,000, ABclonal), GAPDH (AC033, 1:50,000, ABclonal), Lin28B (ab191881, 1:2,000, abcam), rabbit secondary antibody (AS014, 1:10,000, ABclonal), and murine secondary antibody (AS003, 1:10,000, ABclonal).