Af6139

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.

 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.

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.

The wild Cordyceps were collected from Kang ding, Sichuan Province, China. H22 hepatoma cells were kindly provided by Professor Zhang Tian’e (Chengdu University of Traditional Chinese Medicine, Sichuan). Standards of monosaccharides were provided by Sigma (Saint-Louis, MO, USA). Standards of Pullulan (Narrow MWD) were purchased from Shodex, Yokohama, Japan. The 5-fluorouracil (5-FU) for injection was purchased from Abmole Bioscience (Shanghai, China). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were purchased from Chengdu Illio Technology Co., Ltd. (Chengdu, China). Rabbit polyclonal antibodies against B-cell lymphoma-2 (Bcl-2, AF6139), Cytochrome C (Cyto-c, AF0146), and Caspase3 (AF6311) antibody were purchased from Affinity Biosciences (Nanjing, China) and BCL-2-associated X protein (Bax, BA0315-2) antibody was purchased from Boster Biological Technology Co., Ltd. (Wuhan, China). Rabbit monoclonal antibodies against Caspase8 (PTM-6085) antibody were purchased from PTM Biolab Co., Ltd. (Hangzhou, China), and signal transducer and activator of transcription3 (Stat3, BM4052), phosphorylated signal transducer and activator of transcription3 (p-STAT3 Y705, BM4835) were purchased from Boster Biological Technology Co., Ltd. (Wuhan, China). All other chemical reagents were of analytical grade.