Fig. 6: Grail influences sirtuin 1 (Sirt1) protein level and activity.
From: E3 ubiquitin ligase Grail promotes hepatic steatosis through Sirt1 inhibition
A, B Immunoblotting measurement of Sirt1 expression in the liver samples obtained from wild-type (WT), Grail knockout (KO), AAV8-vector, and AAVV8-Grail mice fed high-fat diet (HFD) for 8 weeks. C, D Endogenous Sirt1 protein levels in indicated stable cell lines with or without palmitic acid (PA) treatment. E Effects of WT or mutant GRAIL overexpression on Sirt1 degradation. HEK293 cells were transfected with 0.5 μg of vector, Grail or Grail (H2N2) in the presence of 0.5 μg of pcDNA-flag-Sirt1. The cells were then harvested and subjected to immunoblotting. F, G HepG2/vector and HepG2/Grail cells were added with cycloheximide (CHX, 100 g/ml), and cells were harvested at the indicated times. Cells lysates were subjected to immunoblotting with the indicated antibodies. H, I Endogenous Sirt1 activity was measured in isolated nuclear extracts from HepG2 (GRAIL overexpression or silencing) stable cell lines. Sirt1 activity was expressed as a percentage of the control activity. J, K Oil Red O staining in the indicated HepG2 stable cell lines treated with PA for 24 h. The data are presented as mean ± SD. Student’s t-test are used to evaluate the statistical significance. **P < 0.01; ***P < 0.001.
