{"title":"用腺病毒载体在HepG2细胞中表达大鼠Cyp27b1及其在自制和市售抗Cyp27b1抗体评价中的应用","authors":"Chika Nagao, Satoko Kise, Ayano Iijima, Tadashi Okada, Tomoko Nakanishi, Shigeto Sato, Miyu Nishikawa, Shinchi Ikushiro, Kaori Yasuda, Toshiyuki Sakaki","doi":"10.3177/jnsv.69.90","DOIUrl":null,"url":null,"abstract":"<p><p>Rat Cyp27b1 was successfully expressed in HepG2 cells using an adenovirus vector. High vitamin D 1α-hydroxylation activity was detected in them, whereas no activity was observed in non-infected cells. Similarly, vitamin D 1α-hydroxylation activity was also observed in HepG2 cells expressing Cyp27b1-Flag, which is tagged with a Flag at the C-terminus of Cyp27b1. Western blot analysis using an anti-Flag antibody showed a clear band of Cyp27b1-Flag. Next, we screened three types of anti-Cyp27b1 antibodies, which consist of two commercially available antibodies and our self-made antibody using Cyp27b1- or Cyp27b1-Flag expressing HepG2 cell lysate as a positive control. Surprisingly, Western blot analysis revealed that two commercially available antibodies did not detect Cyp27b1 but specifically detect other proteins. In contrast, our self-made antisera specifically detected Cyp27b1 and Cyp27b1-Flag in the HepG2 cells expressing Cyp27b1 or Cyp27b1-Flag. These commercially available antibodies have been used for the detection of Cyp27b1 by Western blotting and immunohistochemistry. Our results suggest that those data should be reanalyzed.</p>","PeriodicalId":16624,"journal":{"name":"Journal of nutritional science and vitaminology","volume":null,"pages":null},"PeriodicalIF":0.7000,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Expression of Rat Cyp27b1 in HepG2 Cells Using Adenovirus Vector and Its Application to Evaluation of Self-Made and Commercially Available Anti-Cyp27b1 Antibodies.\",\"authors\":\"Chika Nagao, Satoko Kise, Ayano Iijima, Tadashi Okada, Tomoko Nakanishi, Shigeto Sato, Miyu Nishikawa, Shinchi Ikushiro, Kaori Yasuda, Toshiyuki Sakaki\",\"doi\":\"10.3177/jnsv.69.90\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Rat Cyp27b1 was successfully expressed in HepG2 cells using an adenovirus vector. High vitamin D 1α-hydroxylation activity was detected in them, whereas no activity was observed in non-infected cells. Similarly, vitamin D 1α-hydroxylation activity was also observed in HepG2 cells expressing Cyp27b1-Flag, which is tagged with a Flag at the C-terminus of Cyp27b1. Western blot analysis using an anti-Flag antibody showed a clear band of Cyp27b1-Flag. Next, we screened three types of anti-Cyp27b1 antibodies, which consist of two commercially available antibodies and our self-made antibody using Cyp27b1- or Cyp27b1-Flag expressing HepG2 cell lysate as a positive control. Surprisingly, Western blot analysis revealed that two commercially available antibodies did not detect Cyp27b1 but specifically detect other proteins. In contrast, our self-made antisera specifically detected Cyp27b1 and Cyp27b1-Flag in the HepG2 cells expressing Cyp27b1 or Cyp27b1-Flag. These commercially available antibodies have been used for the detection of Cyp27b1 by Western blotting and immunohistochemistry. Our results suggest that those data should be reanalyzed.</p>\",\"PeriodicalId\":16624,\"journal\":{\"name\":\"Journal of nutritional science and vitaminology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.7000,\"publicationDate\":\"2023-01-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of nutritional science and vitaminology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.3177/jnsv.69.90\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"NUTRITION & DIETETICS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of nutritional science and vitaminology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.3177/jnsv.69.90","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"NUTRITION & DIETETICS","Score":null,"Total":0}
Expression of Rat Cyp27b1 in HepG2 Cells Using Adenovirus Vector and Its Application to Evaluation of Self-Made and Commercially Available Anti-Cyp27b1 Antibodies.
Rat Cyp27b1 was successfully expressed in HepG2 cells using an adenovirus vector. High vitamin D 1α-hydroxylation activity was detected in them, whereas no activity was observed in non-infected cells. Similarly, vitamin D 1α-hydroxylation activity was also observed in HepG2 cells expressing Cyp27b1-Flag, which is tagged with a Flag at the C-terminus of Cyp27b1. Western blot analysis using an anti-Flag antibody showed a clear band of Cyp27b1-Flag. Next, we screened three types of anti-Cyp27b1 antibodies, which consist of two commercially available antibodies and our self-made antibody using Cyp27b1- or Cyp27b1-Flag expressing HepG2 cell lysate as a positive control. Surprisingly, Western blot analysis revealed that two commercially available antibodies did not detect Cyp27b1 but specifically detect other proteins. In contrast, our self-made antisera specifically detected Cyp27b1 and Cyp27b1-Flag in the HepG2 cells expressing Cyp27b1 or Cyp27b1-Flag. These commercially available antibodies have been used for the detection of Cyp27b1 by Western blotting and immunohistochemistry. Our results suggest that those data should be reanalyzed.
期刊介绍:
The Journal of Nutritional Science and Vitaminology is an international medium publishing in English of original work in all branches of nutritional science, food science and vitaminology from any country.
Manuscripts submitted for publication should be as concise as possible and must be based on the results of original research or of original interpretation of existing knowledge not previously published. Although data may have been reported, in part, in preliminary or
abstract form, a full report of such research is unacceptable if it has been or will be submitted for consideration by another journal.