Genegnome machine

The expressions of insulin, GLUT2, PGC-1α, GCK, and G6Pase were determined by western blot and GAPDH was employed as control. Pancreas was lysed using cell lysis reagent (Sigma) and phosphatase inhibitors (Sigma) and lysed by Tissue Homogenizer (Bertin Technologies, France). The crude lysate was transferred to new Eppendorf tubes. Each sample was added to 20 μL 2x sample loading buffer (0.125 M of 5 M Tris-HCl, Amresco; 20% glycerol, Usb; 4% of 10% sodium dodecyl sulfate, Amresco; 1%  β-mercaptoethanol, Amresco; 0.2% of 0.05% (w/v) bromophenol blue, Sigma) and boiled for 5 min before loading. Proteins were separated by SDS-PAGE, transferred to immobilon P membrane (Millipore), and probed with different antibodies as indicated. All antibodies were purchased from Cell Signaling (Beverly, MA). The results were visualized using ECL kit (Abcam) and observed by GeneGnome machine (Syngene).

The cells were lysed with RIPA and centrifuged. Protein concentrations were detected using the BCA protein assay kit. The protein was fractionated using SDS-PAGE and transferred onto PVDF membrane. Before incubating with antibodies, membranes were first blocked for in 5% skim- milk. Then the membranes were incubated with primary antibodies at 4 °C overnight, followed incubated with HRP-conjugated IgG antibody at room temperature for 1 h. Finally, the protein was visualized using the ECL kit and observed by the GeneGnome machine (Syngene).