Expression of Rat Cyp27b1 in HepG2 Cells Using Adenovirus Vector and Its Application to Evaluation of Self-Made and Commercially Available Anti-Cyp27b1 Antibodies.
J Nutr Sci Vitaminol (Tokyo)
; 69(2): 90-97, 2023.
Article
en En
| MEDLINE
| ID: mdl-37121728
ABSTRACT
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Vitamina D
/
25-Hidroxivitamina D3 1-alfa-Hidroxilasa
Límite:
Animals
/
Humans
Idioma:
En
Revista:
J Nutr Sci Vitaminol (Tokyo)
Año:
2023
Tipo del documento:
Article