Bcl 2 associated x protein bax

After the treated C28/I2 cells were harvested, 30 μg of protein was extracted by RIPA lysis buffer (Beyotime, Shanghai, China), separated on SDS-PAGE gel and then transferred to polyvinylidene difluoride (PVDF, Millipore, Bedford, MA, USA) membranes. Subsequently, the membranes were immersed in 5% nonfat milk for 2 h and incubated with primary antibodies against B cell lymphoma-2 (Bcl-2, 1:1,000, Abcam, Cambridge, MA, USA), Bcl-2-associated X protein (Bax, 1:2,000, Abcam), SOX11 (1:2,000, Abcam), phosphorylation-P65 (p-P65, 1:1,000, Thermo Fisher Scientific), P65 (5 µg/mL, Thermo Fisher Scientific), IkB-α (1:2,000, Abcam), p-IkB-α (1:3,000, Abcam), Toll-like receptor 4 (TLR4, 1:1,000, Abcam), or GAPDH (1:3,000, Abcam) at 4°C overnight. The membranes were then probed with a secondary antibody conjugated with horseradish peroxidase (1:5,000, Abcam) for 1 h at 37°C. The blots were visualized by RapidStep ECL Reagent (Millipore Corp., Billerica, MA, United States), and the results were observed using Bio-Rad ChemiDoc XRS + Chemiluminescence Imaging System.

OGCs in different groups were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer (Solarbio) containing 1 mM phenylmethanesulfonyl fluoride (PMSF) (Solarbio) at 4 °C for 30 min. The protein concentration was determined with a BCA Protein Assay Kit. Up to 50 µg of protein was electrophoresed on 12% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore), which were blocked with 5% nonfat dry milk (BD Biosciences) for 1 h at room temperature. The membranes were then blotted with primary antibodies at 4 °C overnight. The following primary antibodies were used: β-actin (1:1,000, Proteintech, Rosemont, IL, USA), B cell lymphoma 2 protein (Bcl-2, 1:1,000, Cell Signaling Technology), Bcl-2-associated X protein (Bax, 1:2,000, Abcam), caspase-3 (1:1000, Cell Signaling Technology), cleaved caspase-3 (1:1,000, Cell Signaling Technology) and cleaved poly-ADP-ribose polymerase (cleaved PARP, 1:250, Abcam). Then, the PVDF membrane was washed 3 times with Tris-buffered saline/Tween (Solarbio) and incubated with HRP-conjugated secondary antibody (1:10,000, ZSGB-BIO) for 70 min at room temperature. Detection was performed using Luminata western HRP substrate (Millipore). The results were obtained with a LI-COR 3600 instrument (LI-COR Biosciences, Lincoln, NE, USA) and analysed with an Image Studio Digits Version 4.0 system; n = 5.

Mouse brains were obtained under deep anesthesia 1 and 3 days after surgery, and the ipsilateral hemisphere was used for Western blot analysis. For cells and brain tissue, the lysates were prepared using RIPA lysis buffer (Millipore) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). The Western blot protocol was performed as previously described [21 (link)]. The primary antibodies used were iNOS (1:500; Abcam), Arginase-1(Arg-1; 1:500; Santa Cruz), CD206 (1:800, Abcam), LC3B (1:1000, Sigma-Aldrich), LAMP2 (1:500; Millipore), phospho-mTOR (Ser 2448) (p-mTOR; 1:500; Cell Signaling Technology, Beverly, MA, USA), mTOR (1:1000, Cell Signaling Technology), FLAG (1:1000; Abcam), caspase3 (cas3; 1:500, Cell Signaling Technology), BCL2-associated X protein (bax; 1:1000; Abcam), B cell lymphoma 2 (bcl2;1:500; Cell Signaling Technology), GAPDH (1:1000; Santa Cruz), and β-actin (1:1000; Santa Cruz). The immunoblots were detected using an enhanced chemiluminescence kit (FD Technology, Shanghai, China) and obtained using an imaging system (Bio-Rad, Hercules, CA, USA) and then analyzed by ImageJ software.

DOX was purchased from Haizheng Pfizer Pharmaceutical Co., Ltd. Levosimendan was obtained from Orion Corporation, Espoo, Finland. The following primary antibodies were purchased from Cell Signaling Technology (Boston, MA, USA): Bcl-2-associated X protein (BAX; 1 : 1000), c-caspase-3 (1 : 1000), PTEN (1 : 1000), P-Akt (1 : 1000), T-Akt (1 : 1000), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1 : 1000), and B-cell lymphoma-2 (Bcl-2) (1 : 1000) was purchased from Abcam. Akt inhibitor (Akt i) was purchased from Sigma-Aldrich (St. Louis, MO, USA). A goat anti-rabbit secondary antibody was purchased from LI-COR Biosciences (Lincoln, USA). The BCA protein assay kit was obtained from Dōjindo Laboratories (Kumamoto, Japan).