Fig. 3
From: Sirt2 facilitates hepatic glucose uptake by deacetylating glucokinase regulatory protein
Sirt2 regulates glucokinase and GKRP dissociation. a–c Immunoblot analysis (IB) with the indicated antibodies of total cell lysate and precipitates after immunoprecipitation (IP) with anti-DYKDDDDK (Flag) tag antibodies from Sirt2-knockdown primary hepatocytes transfected with adenovirus vector encoding Flag-tagged glucokinase (Flag-GK) cultured in low- or high-glucose medium (a), from db/db mouse-derived hepatocytes transfected with adenovirus vectors encoding Flag-GK and HA-tagged Sirt2 (HA-Sirt2) cultured in low- or high-glucose medium (b), and from db/db mouse-derived primary hepatocytes transfected with adenovirus vectors encoding Flag-GK with and without Sirt2 knockdown cultured in high-glucose medium with and without NMN (c). GKRP levels coimmunoprecipitated with Flag-GK were normalized to Flag-GK expression and are shown in the right graph of each figure (n = 3 each). d Effect of Sirt2 knockdown on immunoprecipitation and immunoblot analysis of GK−GKRP binding in the liver from mice transfected with adenovirus vectors encoding Flag-GK 30 min after administration of glucose (4 g/kg) after 16-h fasting. e The interaction between Sirt2 and GKRP or glucokinase in primary hepatocytes detected by a fluorescent-based system for detecting protein−protein interactions using hAG-tagged Sirt2, hAG-tagged GKRP, hAG-tagged GK, Ash-Sirt2, Ash-GKRP, and Ash-GK. Scale bars indicate 20 μm. f Immunoprecipitation and immunoblot analysis of the interaction between Sirt2 and GKRP or Sirt2 and GK in HEK293 cells expressing Flag-tagged Sirt2, Halo-tagged GKRP, or Halo-tagged GK. g Immunoprecipitation and immunoblot analysis of GK−GKRP or Sirt2−GKRP binding in mouse primary hepatocytes transfected with adenovirus encoding Flag-GKRP and HA-GK or HA-Sirt2 in low- or high-glucose medium. Data are normalized to control (a−c). *P < 0.05; one-way ANOVA with the Fisher’s PLSD post-hoc test (a–c). All data are representative of at least three independent experiments. Error bars show s.e.m.
