Bm1623

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.

HCC cells were lysed with a strong radioimmunoprecipitation assay buffer containing Halt^TM Protease Inhibitor Cocktail (Thermo, Waltham, MA, USA). The concentrations of the proteins in the lysate were determined with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). Proteins were detected on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane. The membranes were incubated overnight at 4° C with primary antibodies for retinoblastoma (phospho S780) (ab47763, Abcam, Cambridge, UK), retinoblastoma (ab181616) and GAPDH (BM1623, Boster, Wuhan, China), and then were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 2 hours. The proteins were detected using an Amersham Imager 600 (GE Healthcare Life Sciences, Boston, MA, USA).

Cells were lysed in a buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% NP40 (Beyotime, Beijing, China), and protease inhibitors (TaKaRa, Dalian, China). Proteins in lysates were resolved by SDS–PAGE and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA for 1 h at room temperature and incubated with primary antibody recognizing β-catenin (Cell Signaling Technology, Danvers, MA, USA), Lamin B (Cell Signaling Technology, Danvers, MA, USA), or Gapdh (Boster, BM1623, Wuhan, China) at 4 °C overnight. Incubation with secondary horseradish peroxidase-labelled antibody was carried out for 1 h at room temperature. Protein bands were visualized by chemiluminescence using an Electro-Chemi-Luminescence (ECL) kit (Proteintech, Hubei, China) and exposed to X-ray film. Protein band intensities were quantified using Image-J software v. 1.45 (National Institutes of Health, NIH, Bethesda, MD, USA). The GAPDH was used as the internal control for total protein and cytoplasmic protein, and Lamin B1 was used as the internal control for nuclear protein.

Tissues or cells were lysed in in a buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% NP40 (Beyotime, China), and protease inhibitors (TaKaRa, China). Proteins in lysates were resolved by SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA for 1 h at room temperature and incubated with primary antibody recognizing MACF1 (Abcam, ab117418, 1:1000), Runx2 (Abcam, ab23981, 1:500), Osteocalcin (Abcam, ab93876, 1:500), CollagenⅠ (Abcam, ab34710, 1:1,000) , HuR (Abcam, ab200342, 1:1,000), β-Actin (Boster, BM3873, 1:1,000), CBF-A (GeneTex, GTX121922, 1:1,000) , Lamin A/C (Boster, PB0307, 1:1,000) or Gapdh (Boster, BM1623, 1:1,000) at 4 °C. Incubation with secondary horseradish peroxidase-labelled antibody was carried out for 1 h at room temperature.

Cells were lysed in RIPA lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA-Na2, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors and phosphatase inhibitor on ice for 15 min. The cell lysates were cleared by centrifugation at 12,000×g for 10 min, then heat-denatured with 5 ×  Laemmli buffer (G2013, Servicebio). The prepared samples with equal amount of total protein (20 μg) were subjected to SDS-PAGE and immunoblotting, as per the standard procedure. The following antibodies were used: anti-SOX1 (ab109290, Abcam), anti-β-catenin (ab32572, Abcam), anti-HES1 (#11988, Cell Signaling Technology [CST]), anti-PROX1 (11067-2-AP, Proteintech), anti-p38 (#8690, CST), anti-phospho-p38 (#9215, CST), anti-ERK (#4695, CST), anti-phospho-ERK (#4370, CST), anti-AKT (#4691, CST), anti-phospho-AKT (#4060, CST), anti-JNK (#9252, CST), anti-phospho-JNK (#700031, Invitrogen), anti-RAF (#9422, CST), anti-phospho-RAF (#9427, CST), anti-MEK (51080-1-AP, Proteintech), anti-phospho-MEK (#9154, CST), anti-α-Tubulin (11224-1-AP, Proteintech), anti-GAPDH (BM1623, Boster), anti-BCL2 (Proteintech, 12789-1-AP), anti-PCNA (BM0104, Boster). All antibodies were diluted by antibody dilutions (G2025, Servicebio) with a ratio of 1:1,000. All quantitative analyses were performed using the software image J.

Cells were collected and lysed in the whole-cell lysate (containing phenylmethylsulfonyl fluoride and a phosphatase inhibitor). Equal amounts of cell lysates were separated by 10% SDS-PAGE and transferred to PVDF membranes. After blocking in 5% skimmed milk for 1 h at 37°C, membranes were incubated overnight at 4°C with the following primary antibodies: anti-GRP78 (AF5366, Affinity, China, 1 : 1000), anti-PERK (ab229912, Abcam, UK, 1 : 1000), anti-phospho-PERK (p-PERK, DF7576, Affinity, China, 1 : 2000), anti-CHOP (BF8081, Affinity, China, 1 : 1000), anti-programmed cell death receptor 1 (PD-1, ab214421, Abcam, UK, 1 : 1000), and anti-mucin domain 3 (TIM-3, D3M9R, Cell Signaling Pathway, USA, 1 : 1000), and anti-GAPDH (BM1623, Boster Biological Technology, China, 1 : 5000) was used as a control. Following the primary incubation, the membranes were incubated with horseradish peroxidase-conjugated goat antimouse IgG secondary antibody (ZJ2020, Biygot, China, 1 : 10000) at room temperature for 2 h. After washing the membrane with TBST for 45 min, the protein signal was detected using ECL Plus Hypersensitive Luminescence solution (AR1197, Boster, China).

Angiotensin II, chloroquine (CQ), Bafilomycin A1(Baf A1) and 3-methyladenine (3-MA) were purchased from Sigma Aldrich (Sigma Aldrich, USA). Primary antibodies for detecting Sirt3 (rabbit monoclonal) (D22A3) [5490], FoxO1 (rabbit monoclonal) (C29H4) [2880], LC3 (rabbit polyclonal) [2775], LC3 (rabbit polyclonal) [3868], Beclin-1 (rabbit monoclonal) (D40C5) [3495] were purchased from Cell signalling Technology (CST, UK). Primary antibodies against ac-FoxO1 (rabbit polyclonal) (FKHR D19) [sc49437], MuRF1 (mouse monoclonal) [sc398608], MAFBx (mouse monoclonal) (sc166806) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Primary antibodies against p62 (mouse monoclonal) [ab56416] was obtained from Abcam. Primary antibodies against β-Tubulin (mouse monoclonal) [BM1453], GAPDH (mouse monoclonal) [BM1623], α-SMA (mouse monoclonal) [BM0002] was purchased from Boster.