Quantification of Notch1 and NICD1 Proteins

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).