title: Identification of AFB1-interacting proteins and interactions between RPSA and AFB1.
abstract: A method using immobilized affinity chromatography (IAC) was developed to screen for aflatoxin B1 (AFB1)-binding proteins. AFB1 and bovine serum albumin (BSA) coupled protein (BSA-AFB1) was prepared using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The resulting coupled compound was immobilized onto PVDF transfer membranes, which were then incubated with total protein from mouse liver. AFB1-binding proteins were eluted, after non-specific washing, by specific elution, and the eluted proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two candidate AFB1-binding proteins were identified by liquid chromatography-tandem mass spectrometry as the 40S ribosomal protein SA (RPSA) and a putative uncharacterized protein. RPSA and AFB1 interactions were further analyzed by ELISA in vitro and laser confocal immunofluorescence analysis in vivo. The results from ELISA and immunofluorescence showed that RPSA efficiently bound AFB1 in vitro and in vivo. This study's conclusion laid the foundation for further exploration of the role of AFB1-binding proteins in AFB1 toxicology towards hepatocytes and the entry pathway of AFB1 into hepatocytes.
authors.affiliation: Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China. Electronic address: xzhzhenhong@163.com.
authors.forename: Zhenhong
authors.initials: Z
authors.lastname: Zhuang
authors.affiliation: Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
authors.forename: Yaling
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China. Electronic address: 1094324057@qq.com.
authors.forename: Shihua
authors.initials: S
authors.lastname: Wang
mesh_terms: D016604:Aflatoxin B1 / Q000378:metabolism*; D000818:Animals; D045744:Cell Line, Tumor; D002470:Cell Survival; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D051379:Mice; D017474:Receptors, Laminin / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D012709:Serum Albumin / Q000378:metabolism*
chemical_list: D055767:Immobilized Proteins; D017474:Receptors, Laminin; D012269:Ribosomal Proteins; C000609616:Rpsa protein, mouse; D012709:Serum Albumin; C052669:aflatoxin B1-bovine serum albumin; D016604:Aflatoxin B1
keywords: AFB1-binding proteins; ELISA; IAC; Immunofluorescence; RPSA