Anti g6pase

Expression of the enzymes involved in glucose metabolism and in cytosolic and ER PPP was determined by Western blot, using standard procedures. Anterior and posterior brain regions were homogenized in PBS in the presence of a protease inhibitor cocktail for mammalian cells, and total protein was measured by Bradford assay. After SDS-PAGE, performed according to the standard method on 10% precast gels (BioRad), proteins were transferred to a nitrocellulose membrane. The membrane was blocked for 1 h with Tris Buffered Saline (TBS) plus 0.15% Tween 20 (TBSt) containing 5% nonfat dry milk and incubated overnight at 4 °C with the following rabbit polyclonal antibodies: anti-HK II (1:1000, Cell Signalling #2867), anti-G6Pase (1:1000, Abcam, ab93857), anti-PFKP (1:200, Cell Signalling #8164), anti-G6PD (1:1000, Abcam, ab210702), anti_H6PD (1:1000, Abcam, ab170895), or anti-actin (1:10,000, Thermo-Fisher MA5-11869). After washing with TBSt, the membrane was incubated with an anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP) (BioRad) and developed with Clarity Western ECL Substrate (BioRad). Bands were detected and analyzed for density using an enhanced chemiluminescence system (Alliance 6.7 WL 20 M, UVITEC, Cambridge, UK) and UV1D software (UVITEC). Bands of interest were normalized for actin levels in the same membrane.

Phloroglucinol and glucagon were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in 0.1% DMSO and distilled water, respectively. Antibodies against AMPKα and phospho-AMPKα (Thr-172) were purchased from Cell Signaling Technology (Beverly, MA, USA) and anti-PEPCK and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-G6Pase was purchased from Abcam (Cambridge, MA, USA). Compound C and glucose-free DMEM were purchased from Sigma-Aldrich (St. Louis, MO, USA).