Protocol for Assessing Metabolic Pathways

Dulbecco's Modified Eagle's Medium (DMEM), trypsin-EDTA, penicillin-streptomycin solution, and fetal bovine serum (FBS) were acquired from Hyclone (South Logan, UT, USA). Antibodies against NKAα1, NKAα2, NKAα3, P110α, P110β, PEPCK, G6pase, β-actin, β-tubulin, GAPDH, and the secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alexa Fluor 568 conjugated goat anti-rat IgG (H+L) was purchased from Invitrogen Corporation (Carlsbad, USA). Antibodies against pan-cadherin, phospho-GS (Ser641), phospho-GSK3 (Ser9), GS and GSK3 were obtained from Abcam (Cambridge, MA, USA). The generation of a DR-region-specific antibody (⁸⁹⁷DVEDSYGQQWTYEQR⁹¹¹) was performed as our previous reports 32 (link), 33 (link). Hematoxylin and eosin (H&E) staining kit, G6pase and PEPCK activity kits, periodic acid-schiff (PAS) kit, glucose assay kit, insulin assay kit, and glycogen assay kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The specific primers were provided by Integrated DNA Technologies Pte. Ltd. (Singapore). LY294002 and MK2206 were bought from Selleck Chemicals (Houston, TX, USA). D12492 60 kcal% fat was obtained from Research Diets (New Brunswick, NJ, USA). Glucosamine (GlcN), insulin, glucose, and phosphate colorimetric kit were obtained from Sigma (St. Louis, USA).