Bcl2 associated x (bax)

[d-Ala2, d-Leu5]-Enkephalin (DADLE, a selective DOR agonist), 3-methyladenine (3-MA, an autophagy inhibitor) and antibody against LC3 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Naltrindole (a DOR antagonist) was obtained from Tocris (Ellisville, MO, USA). DMEM, FBS, 0.05% Trypsin-EDTA and normal goat serum were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Cell Counting Kit-8 (CCK-8) kit, LDH kit, BCA kit, Cyanine 3 (Cy3)-conjugated secondary anti-rabbit antibodies, Alexa Fluor 488 goat anti-mouse IgG and Hoechst were obtained from Beyotime (Nantong, Jiangsu, China). PMSF (a protease inhibitor cocktail and a phosphatase inhibitor cocktail) was obtained from KangChen (Shanghai, China). Antibodies against beclin 1, p62 and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against glial fibrillary acidic protein (GFAP), Bcl-2, Bax and an anti-rabbit IgG antibody linked with HRP were obtained from Bioworld (Shanghai, China). An ECL kit was obtained from Millipore (Billerica, MA, USA).

The right hippocampus was assigned to detect the protein expression of Caspase-3 (CASP3), B-cell lymphoma 2 (BCL2), and BCL2-associated X protein (BAX) using Western blotting assays (n=6 for each group). For protein extraction, tissue was collected and added to RIPA buffer with 1% PMSF and then homogenized in lysis buffer on ice. Total proteins were extracted following the manufacturer’s instructions (Applygen Technologies, Inc., China). After determining protein concentrations, samples (30–50 μg) were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and subsequently transferred to immobile polyvinylidene difluoride membranes. Nonspecific bindings were blocked with 5% nonfat milk in TBST buffer (pH=7.4) for 1 h, then the membranes were incubated with the following primary antibodies overnight at 4°C: anti-CASP3 (1: 500; Bioworld, USA), BCL2 (1: 500; Bioworld, USA), BAX (1: 500; Bioworld, USA), and anti-β-actin (1: 1000; Santa Cruz, CA, USA). Subsequently, after washing with 0.1% TBST, membranes were incubated with secondary antibody (1: 4000; Rockland, Gilbertsville) for 1 h at room temperature and washed in 0.1% TBST solution. Bands were detected using the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA) and the relative density of bands was estimated using Image J analysis software.

After cells were treated with different conditions, they were lysed in a lysis buffer including protease inhibitors. Protein concentrations were measured using a BCA kit. Twenty micrograms of protein per sample was separated on a 12% SDS gel, followed by semi-dry western blotting and transfer onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% BSA in TBST [50 mM Tris–HCl (pH 7.5) and 150 mM NaCl containing 0.05% Tween 20] (24 (link)) for 1 h, and incubated overnight at 4 °C with primary antibodies against β-actin (1:1,000, rabbit, HuaBio), LC3B (1:1,000, rabbit, Bioworld), Beclin1 (1:1,000, mouse, Bioworld), SQSTM1/P62 (1:1,000, rabbit, CST), Bcl2 (1:1,000, rabbit, CST), cleaved caspase3 (1:1,000, rabbit, CST), Bax (1:1,000, rabbit, Bioworld), Erk1/2 (1:1,000, rabbit, CST), and p-Erk1/2 (1:1,000, rabbit, CST). Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies (1:10,000 dilution, anti-rabbit or anti-mouse, CST) for 1 h at room temperature. Bands on the immunoblots were visualized with Millipore Immobilon ECL (Millipore, USA) and detected on Bio-Rad ChemiDoc™ XRS+.

RIPA buffer containing phosphatase and protease inhibitors was used to extract proteins from samples. The concentration of protein was measured with a BCA kit (Thermo Fisher Scientific, Rockford, IL, USA). The proteins were separated by SDS‒PAGE and then transferred onto PVDF membranes. After being blocked, the membranes were incubated with primary antibodies against Bax (Bioworld, Minneapoils, MN, USA, BS2538), Bcl-2 (Bioworld, BS1511), iNOS (BD Biosciences, NJ, USA, 610328), MCP-1 (Proteintech, Wuhan, China, 66272-1-Ig), TNF-α (Proteintech, 17590-1-AP), β-actin (Bioworld, AP0060), lamin B1 (Bioworld, AP6001), HMGB1 (Abcam, Cambridge, MA, USA, ab18256), GAPDH (Bioworld, AP0063), acetyl-lysine (PTM-BioLab, Hangzhou, China, PTM-105) and HSP90AA1 (Invitrogen, PA3-013) overnight at 4°C. Next, after the membranes were incubated with HRP-conjugated secondary antibodies and washed with TBST, the bands were visualized by a GelPro system (Tanon Technologies, Shanghai, China) using an ECL Detection Kit (Millipore, Billerica, MA, USA), and the band intensities were analyzed by ImageJ software.