Ba0315 2

After isolation from tissue or cell samples, total proteins were separated via SDS-PAGE and transferred onto PVDF membranes that were blocked with an appropriate solution for 1 h at room temperature. Blots were then probed for 2 h at room temperature with primary antibodies specific for Bax (1:1000, BA0315-2, Boster), Bcl-2 (1:1200, A00040-1, Boster), Cytochrome C (1:5000, ab133504, Abcam), DNAJB4 (0.1 µg/ml, ab254641, Abcam), and GAPDH (1:10,000, BA2913, Boster). Blots were then probed for 1 h at room temperature using Goat Anti-Rabbit IgG H&L (HRP) (1: 2000, ab6721, Abcam), after which they were washed, incubated in developer solution, and exposed to X-ray film to detect protein bands.

Total protein was extracted from the frozen thoracic aorta of rats and HUVECs with RIPA lysis buffer containing protease inhibitor cocktail. The protein concentrations were determined by the Bradford protein assay kit. Equal amounts of protein lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes followed by a block with 5% skimmed milk at 4°C for 1.5 h. Subsequently, the membranes were incubated with primary antibodies against eNOS (AF0096, Affinity, 1:1,000) and p-eNOS (AF3247,Affinity, 1:1,000), c-jun N-terminal kinase (JNK BS1544, Bioworld, 1:500) and p-JNK (BS4322, Bioworld, 1:500), B-cell lymphoma-2 (Bcl-2, BA0412, Boster, 1:800), BCL2-Associated X (Bax, BA0315-2, Boster, 1:800), inositol-requiring enzyme 1α (IRE1α, bs-8680R, Bioss, 1:1,000), glucose regulated protein 78 (GRP78, ab21685, Abcam, 1:1,000), CHOP, respectively. β-actin (M02014-5, Boster, 1:1,500) was used as the internal reference. Antibody binding was detected with enhanced chemiluminescent agent and quantified with Image J software. Each experiment was repeated three times.

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.