Protease inhibitors

Total proteins were extracted using radio immunoprecipitation assay (RIPA) buffer with protease inhibitors (Boston Bio Products, Boston, MA, USA). Plasma membrane (PM) proteins were extracted using the Membrane Protein Extraction Kit (BestBio, Shanghai, China). Protein levels were quantified by the BCA protein assay. Total protein (25 μg) was loaded onto a 10% SDS-PAGE gel, separated by electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane. Blots were blocked with 6% skim milk and incubated overnight at 4 °C with primary antibody against GLUT4 (Abcam, Cambridge, UK)and IRS1 (Proteintech Group, Chicago, IL, USA), followed by incubation with secondary antibody for 1 h at room temperature and measured with a Fluor Chem M (ProteinSimple, Santa Clara, CA, USA). Protein expression was normalized by detection of β-actin (Abcam, Cambridge, UK). Duplicate experiments were conducted for all primary adipocyte proteins. The data were analyzed by Image J software (National Institutes of Health, Bethesda, MD, USA) and expressed as fold-change relative to the control group after normalizing against β-actin. Statistical differences between treatment and control groups were determined using Student’s t-test at p < 0.05.

Hepatic homogenates were prepared by homogenizing frozen livers in cold RIPA buffer containing protease inhibitors (Boston Bio Products, MA). Hepatic protein (20 μg) was subjected to NuPAGE 5–10% Bis-Tris gel electrophoresis and transferred to polyvinylidene fluoride membranes (0.45 μm), which were subsequently blocked with 5% milk, incubated overnight at 4 °C with primary antibodies against CPS1 (Santa Cruz Biotechnology, Santa Cruz, CA) and SIRT5 (Cell Signaling Technology, Inc., Danvers, USA) followed by secondary antibodies for 1 h at room temperature. β-actin (Abcam, Cambridge, UK) was used as the control. Duplicate experiments were carried out for all hepatic proteins. The blot was scanned in a FluorChem M (ProteinSimple, Santa Clara, California, USA). The data were analysed by Image J software and expressed as fold-change relative to the control group after normalizing against β-actin.