Radioimmunoprecipitation assay ripa buffer

The seaweed Dictyopteris divaricata, was purchased in the form of dry powder from Dalian, Liaoning, China. The stool DNA isolation kit (FORGENE) was obtained from Chengdu, China. The bicinchoninic acid (BCA) protein kit and serum insulin, interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and Malondialdehyde (MDA): Mouse ELISA kits were purchased from TransGen Biotech Co, Ltd., Beijing, China, and Shanghai Longton Biotechnology Co, Ltd., Shanghai, respectively. Serum Super Oxide Dismutase (SOD) kit was purchased from Solarbio Life Sciences (catalog no. BC0170). The primary antibodies [goat antirabbit: β-actin, Mucin-2 (MUC-2), Claudin-1, Occludin, Zonula occludens-1 (ZO-1) and insulin receptor substrate-1 (IRS-1)], secondary antibodies and the radioimmunoprecipitation assay (RIPA) buffer were purchased from Proteintech (Wuhan, China). The DAB substrate chromogen system and horseradish peroxidase-conjugated secondary antibody were from ZSGB-BIO (Beijing, China). Streptozotocin (STZ) was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and stored at -20 ℃ freezer. All the other chemicals used in the experiments were of highest analytical grade and purchased commercially.

The fruiting body of the mushroom Dictyophora indusiata was obtained from Anhui Joy Lok Food Co., Ltd., Ningde, Fujian Province, China. The broad-spectrum antibiotics clindamycin hydrochloride and metronidazole were from Tian Fang Pharmaceutical Co., Ltd. (Zhumadian, China) and Changchun Wan de Pharmaceutical Co., Ltd. (Changchun, China) respectively. The DNA extraction kit (QIAamp DNA Stool Mini Kit) and gel purification kit (Agencourt AMPure XP 60 mL Kit) were from Qiagen (Hilden, Germany) and Beckman Coulter (Brea, CA, USA), respectively. The primary antibodies (β-actin, claudin-1, occludin, and zonula occludens-1 (ZO-1)), secondary antibodies, and the Radioimmunoprecipitation assay (RIPA) buffer were from Proteintech (Wuhan, China). The Mucin-2 (MUC2) primary antibody was from Boster Biological Technology, Wuhan, China. The horseradish peroxidase-conjugated secondary antibody and DAB substrate chromogen system were from ZSGB-BIO (Beijing, China). The bicinchoninic acid (BCA) protein assay kit was from Pierce Rockford, IL, USA. The Limulus amebocyte lysate (LAL) assay kit was obtained from Chinese horseshoe crab reagent manufactory Co., Ltd. (Xiamen, China). The ELISA kits were purchased from Shang Hai Lengton Bioscience Co, Ltd., Shanghai, China. All the other chemicals used in this study were of analytical grade and purchased from the standard commercial sources.

The mushroom M. esculenta fruiting body was bought from Shandong Tai’an Yinsheng Food Co., Ltd., Shandong, China. Streptozotocin (STZ) was purchased from Sigma Chemical Co. (St. Louis, MO). High-fat diet (HFD) (45% fat Kcal%) was purchased from MediScience Ltd. (Yangzhou, China). The stool DNA isolation kit (FORGENE) was provided by Chengdu, China, and the gel purification kit (Agencourt AMPure XP 60 mL Kit) was obtained from Beckman Coulter (Brea, CA, USA). The bicinchoninic acid (BCA) protein assay kits from TransGen Biotech Co., Ltd., Beijing, China. All ELISA (enzyme-linked immunosorbent assay) kits were purchased from Shanghai Longton Biotechnology Co., Ltd., in Shanghai. The primary antibodies [goat anti-rabbit, β-actin, claudin-1, zonula occludens-1 (ZO-1), occludin and mucin-2 (MUC2)] secondary antibodies, and the Radioimmunoprecipitation assay (RIPA) buffer were purchased from Proteintech (Wuhan, China). All other chemicals were of analytical grade and obtained from standard commercial sources.