Detection of Sirtuin-1 protein expression in peripheral blood leukocytes in dogs

• Published in: Journal of Veterinary Medical Science Vol. 80; no. 7; pp. 1068 - 1076
• Main Authors: YOSHIMURA, Kuniko, MATSUU, Aya, SASAKI, Kai, MOMOI, Yasuyuki
• Format: Journal Article
• Language: English
• Published: Japan JAPANESE SOCIETY OF VETERINARY SCIENCE 2018; Japan Science and Technology Agency; The Japanese Society of Veterinary Science
• Subjects: Adenine; Amino acid sequence; Animals; Blotting, Western; dog; Dogs; Flow cytometry; Histone deacetylase; Humans; Internal Medicine; Leukocytes (granulocytic); Leukocytes (mononuclear); Leukocytes, Mononuclear - metabolism; Life span; Lymphocytes; Monoclonal antibodies; Monocytes; NAD; Nicotinamide; Peripheral blood mononuclear cells; Reverse transcription; SIRT1 protein; Sirtuin 1 - genetics; Sirtuin 1 - metabolism; sirtuin-1; Western blotting; sirtuin-1; dog; flow cytometry
• ISSN: 0916-7250; 1347-7439; 1347-7439
• DOI: 10.1292/jvms.17-0499

Sirtuin-1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase with a large number of protein substrates. It has attracted a lot of attention in association with extending lifespan. The objective of this study was to enable the evaluation of SIRT1 expression in peripheral blood mononuclear cells (PBMCs) from dogs by flow cytometry. Three transcript variants were amplified from PBMCs by reverse transcription PCR and the nucleotide sequences were analyzed. On the basis of deduced amino acid sequence, a monoclonal antibody against human SIRT1, 1F3, was selected to detect canine SIRT1. Canine SIRT1 in peripheral blood mononuclear cells was successfully detected by western blotting using this antibody. Intracellular canine SIRT1 was also detected in permeabilized 293T cells transfected with a canine SIRT1 expression plasmid by flow cytometry using this antibody. SIRT1 was detected in all leukocyte subsets including lymphocytes, granulocytes and monocytes. The expression level was markedly different among individual dogs. These results indicated that the method applied in this study is useful for evaluating canine SIRT1 levels in PBMCs from dogs.