Af0120

Isolated renal tubular cells of rats, various cultured HK-2 cells, and NRK-52E cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Beyotime) and separated by SDS-PAGE. Primary antibodies against CD133 (1:500, Abcam), Vimentin (1:1000, Proteintech), CK-18 (1:1000, Boster), PCNA (1:1000, Proteintech), P53 (1:1000, #2524S, Cell Signaling Technology, MA, USA), Bax (1:1000, AF0120, Affinity), caspase-3 (1:500, ab32351, Abcam), cleaved caspase-3 (1:500, BF0711, Affinity), β-actin (1:5000, TA-09, ZSBIO, Beijing, China) and β-tubulin (1:5000, T0023, Affinity, IL, USA) and conjugated secondary antibodies (1:10,000, ZB-2301, ZB-2305, ZSBIO) were used. All blots were cut prior to hybridization with antibodies during blotting. Signals of targeted proteins were detected by ECL detection Reagent (ED0015-B, Sparkjade, China). Protein expression levels were normalized to those of β-actin or β-tubulin.

Phosphatase inhibitors and protease inhibitors (Millipore, Burlington, MA, USA) were added to SDS lysis buffers to homogenize the hippocampus and cortex. With the Bradford method (Bio-Rad, Hercules, CA, USA), the protein supernatants of the lysates were obtained after centrifugation at 15,000 r and 4 °C for 15 min. Proteins were transferred to PVDF membranes (Millipore) after isolation on SDS-PAGE gels, sealed with skim milk and detected overnight with primary antibody at 4 °C. Overnight incubation occurred of endoplasmic reticulum stress related antibodies, against apoptotic antibodies and neuroinflammation antibodies: PSD95 antibody #ab12093 (abcam, Cambridge, UK); Synaptophysin antibody #ab52636 (abcam); GFAP antibody #95717 (Cell Signaling Technology(CST), Boston, MA, USA); Anti-TNF Receptor I antibody #ab19139 (abcam); Bax antibody #AF0120 (Affinity Biosciences, Cincinnati, OH, USA); Bcl-2 antibody #AF6139 (Affinity Biosciences); β-actin antibody # A1978 (Sigma, Ronkonkoma, NY, USA); Anti-rabbit IgG (CST). Immunoreaction of anti-mouse IgG antibody (CST) was completed, ECL luminescent solution was 1:1, and the immunoblotting was recorded by ChemiDocXRS+ (Bio-RAD, Hercules, CA, USA), a comprehensive gel imager.

The expression of BAX, BCL-2 and HINT1 were evaluated with western blot analysis. Isolation of four encephalic regions was performed on ice. Samples from the MWM subgroups were lysed in RIPA lysis buffer (Beyotime, Guangzhou, China), and the protein concentration was determined with a BCA protein assay kit (Pierce, Rockford, IL, USA). The protein extracts (30 μg per lane) were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes, which were probed with various primary antibodies. After incubation with the appropriate secondary antibodies for 1 h at room temperature, the membranes were treated with ECL reagents (Bio-Rad, Hercules, CA, USA), and the signals were visualized with an Odyssey Imaging System. The expression levels of specific proteins were normalized to those of β-actin. Image Lab software (Bio-Rad) was used for quantification analysis. The following primary antibodies were used: anti-BAX antibody (AF0120, 1:1000, rabbit monoclonal, Affinity Biosciences, Cincinnati, OH, USA), anti-BCL-2 antibody (AF6319, 1:1000, rabbit monoclonal, Affinity Biosciences), HINT1 (ab124912, 1:1000, rabbit monoclonal, Abcam, Cambridge, MA, USA) and β-actin (C4, 1:5000, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

 According to the standard scheme, the expression of Bax and Bcl-2 in myocardial tissue was detected by Western blotting. Protein was extracted from myocardial tissue in a buffer containing RIPA lysate and protease inhibitor. The concentration of the protein was tested by BCA kit. The same amount of protein (30 μg) was transferred from SDS-polyacrylamide gel to polyvinylidene fluoride membrane. PVDF membrane was sealed for 1 h with 5% skim milk powder and incubated with primary antibody anti-Bax (AF0120, 1:2,000 dilution, Affinity, USA), anti-Bcl-2 (AF6139, 1:1,000 dilution, Affinity, United States), or anti-GAPDH (AB0036, 1:5,000 dilution, Abways, Shanghai, China) overnight at 4°C. PVDF membrane was washed 3 times with TBST for 10 min each time and incubated the second antibody labeled with HRP and shaked the bed for 1 h. The protein signal were detected by using enhanced chemiluminescence reagents (ECL) in Chemilluminescence Imaging System (ChemiScope 6,000 system pro, clinx, Shanghai, China) and analyzed by Quantity One software (Bio-Rad, Richmond, California, CA, United States).

The intestinal tissues were made into paraffin section, and dewaxed with xylene, then ethanol was successively added with high to low concentration to rehydrate the tissue, and antigen repair solution was added, then the sections were incubated in 5% BSA (G5001, SERVICEBIO), 0.2% Triton X-100 and PBS (G0002, SERVICEBIO) to permeabilize the tissue sections. Followed, the sections were incubated with the BAX (AF0120, 1 : 200, Affinity) and Bcl-2 (AF6139, 1 : 200, Affinity) overnight at 4°C, then incubated with Goat Anti-Rabbit IgG (H + L) Fluor488-conjugated (S0018, Affinity) at room temperature for 0.5 hours. Then washed away the antibody, and the DAPI (G1012, SERVICEBIO) was added to stain the cell nuclear. The NIKON ECLIPSE C1 was used to observe and take pictures. ImageJ software was used to analyze the BAX and Bcl-2 protein fluorescence intensity.

For Western blots, primary antibodies against bax (AF0120) and caspase-3 (AF6311) were purchased from Affinity Biosciences Inc. (OH, United States). The primary antibody against bcl-2 (2876) was obtained from Cell Signaling Technology, Inc. (MA, United States). The primary antibody against caspase-9 (ab184786) was bought from Abcam (Cambridge, MA, United States), while the anti-cytochrome c antibody (bs-0013R) was purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China). The primary antibody against PARP (AP102), COX IV (AC610), and β-actin (AA128) was obtained from Beyotime (Beijing, China). All the above primary antibodies except β-actin are rabbit polyclonal antibodies while the latter is a mouse monoclonal antibody. Horseradish peroxidase-labeled AffiniPure goat anti-rabbit and anti-mouse secondary antibodies were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). Recombinant murine TNF-α (C600052) was purchased from Sangon Biotech Co., Ltd. (Shanghai, China).

The OC tumor tissue and cells were extracted to obtain the protein, and the protein was quantified by using the BCA kit. The 50 μg protein was loaded quantitatively and separated by SDS-PAGE with 10% separation gel. Electrophoresis conditions were as follows: 80 V (30 min) and then 120 V; electrophoretic transfer condition was as follows: 90 min, 200 mA, and transfer protein to the PVDF membrane; it was then sealed for 2 h in skim milk powder solution; the corresponding primary antibodies anti-NF-κB p65 (ab16502, Abcam, Cambridge, 1 : 500), anti-p38 (Abcam, ab31828, 1 : 1000), anti-mTOR (phospho S2448)(Abcam, ab109268, 1 : 1000), phospho-PI3K p85 (AF3242, Affinity, 1 : 500), anti-AKT (phospho T308) (ab38449, Abcam, 1 : 500), anti-Bcl-2 (Affinity, AF6139, 1 : 1000), anti-Bax (Affinity, AF0120, 1 : 1000), anti-Cyt-C (Affinity, AF0146, 1 : 1000), anti-caspase-3 (Affinity, AF6311, 1 : 1000), anti-cleaved caspase-3 (Affinity, AF7022, 1 : 1000), and anti-GAPDH (ab8245, Abcam, 1 : 500) were added proportionally and incubated overnight at 4°C. After washing the membrane, a second antibody was added and incubated at room temperature for another 1 h. After washing the membrane again, the developer and fixative 1 : 1 were mixed; then, the protein strip was developed with a gel imaging system, and the statistical results were analyzed.