Quantity one software version 4.6.8 for windows

Total proteins were extracted from either rat liver tissue or cells in a lysis buffer. The quantified proteins were separated by electrophoresis in a 10% SDS polyacrylamide gel, as previously described [10 (link),28 (link)]. The proteins were subsequently transferred from the gel onto a nitrocellulose membrane. To determine the relative amount of pAMPK, the membranes were probed with rabbit anti-phospho-AMPK-a monoclonal antibody (1:1000) or rabbit anti-AMPK-a monoclonal antibody (1:1000) that were purchased from Cell Signaling Technology (Danvers, MA, USA). To ensure equal loading of protein quantity in each lane of the gel, the membranes were probed with an antibody against a housekeeping protein, rabbit anti-β-actin monoclonal antibody (1:1000, Cell Signaling Technology). HRP-conjugated anti-rabbit IgG secondary antibodies (1:2000, Cell Signaling Technology, Danvers, MA, USA) were used, and the protein bands were detected using the Enhanced Chemiluminescence detection system Millipore Ltd., Burlington, MA, USA). The amount of protein in each lane was then quantified using Quantity One software version 4.6.8 for Windows (Bio-Rad, Herculesm, CA, USA).

The protein levels of Notch1 and cleaved Notch1/Notch1 intracellular domain (NICD1) were detected using western immunoblotting. Total proteins were extracted from mouse liver tissues in a protein lysis buffer containing 20 mM Tris at pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 2.1 μM leupeptin, 1 mM PMSF and 1% (v/v) Triton X-100. Extracted proteins were quantified and separated in a 10% SDS polyacrylamide gel as previously described [23 (link),24 (link)]. Following electrophoresis and electrotransfer onto nitrocellulose membrane, the blots were probed with rabbit anti-Notch1 monoclonal antibody or rabbit anti-NICD1 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA). The membranes were then reprobed with rabbit anti-β-actin monoclonal antibody (Cell Signaling Technology) to ensure equal loading of the samples. All the blots were incubated with HRP-conjugated anti-rabbit IgG secondary antibodies (Cell Signaling Technology). Proteins were visualized by using an ECL detection system (Bio-Rad, Hercules, CA, USA) and quantified using Quantity One software version 4.6.8 for Windows (Bio-Rad).