Sc 7908

Western blotting was performed as previously described^{30 (link)}. Liver tissues and cultured cells were homogenized in ice-cold CelLytic MT Cell Lysis Reagent (Sigma) with protease inhibitors. Immunoprecipitation was performed using Acetylated K antibodies conjugated to Dynabeads Protein G (Life Technologies) or Flag tag antibody conjugated-magnetic beads. The antibodies used in the immunoprecipitation of Flag tag, acetylated K of GKRP, and acetylated K of keratin 8 were anti-DYKDDDDK tag magnetic beads (1E6, 10 μl/mg protein; Wako), anti-AcK (#9441, 0.2 μl/mg protein; Cell Signaling Technology, Danvers, MA), and anti-AcK (ab21623, 0.2 μl/mg protein; Abcam, Cambridge, UK), respectively. The following antibodies were obtained for immunoblotting: anti-Sirt2 (sc-20966, 1:500), anti-glucokinase (sc-7908, 1:500), anti-GKRP (sc-11416, 1:500) (Santa Cruz Biotechnology), anti-Sirt1 (07-131, 1:1000; Millipore, Billerica, MA), anti-Flag (F1804, 1:1000), anti-β-actin (A5441, 1:1000) (Sigma), anti-HA (3F10, 1:1000; Roche), anti-HAlo (G9281, 1:1000; Promega), and anti-keratin 8 (TROMA-I, 1:200; Developmental Studies Hybridoma Bank, Iowa City, IA). Immunoblot images were quantified by densitometry on an LAS-3000 Imager (Fujifilm, Tokyo, Japan). Uncropped images of representative immunoblots are shown in Supplementary Fig. 9.

The following antibodies and dilutions were used: rabbit anti-GK (1∶100, sc7908; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse anti-vimentin (1∶200, DAKO, Santa Barbara, CA, USA). The antibodies were diluted in a Tris-HCl buffer (pH 7.8) containing 8.4 mM sodium phosphate, 3.5 mM potassium phosphate, 120 mM sodium chloride, and 1% bovine serum albumin. Sections of the liver (20 µm) and frontal hypothalamus (40 µm) were fixed directly by immersion in 4% (w/v) paraformaldehyde for 12 h, incubated in 30% sucrose for 3 days and cut with a cryostat (Leica, microm HM520), and subsequently processed free-floating. Primary antibodies were incubated overnight at 22°C, and subsequently incubated for 2 h at 22°C with Cy²- Cy³-labeled secondary antibodies (1∶200; Jackson ImmunoResearch Laboratories, Inc., Pennsylvania, USA). The samples were counter-stained with the DNA stain, TOPRO-3 (1∶1000; Invitrogen, Rockville, MD, USA), and the slides were analyzed by confocal laser microscopy (Carl Zeiss, LSM700, Jena, Germany). A total of five rats were used per glycemic conditions.