Fusion fx7 image and analytics system

Total protein was extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, MA, USA) and the protein concentration was determined using BCA Protein Assay Kit (Thermo Scientific, MA, USA), according to the manufacturer's protocols. Equal amount of protein was fractionated using SDS-PAGE before transfer to nitrocellulose membranes (Bio-Rad Laboratories, CA, USA). Membranes were blocked with bovine serum albumin (AMRESCO, OH, USA) or non-fat skim milk (Merck, Hesse, Germany) and were then incubated with primary antibodies: β-catenin rabbit monoclonal antibody (1:1000) (Cell Signaling Technology, MA, USA), vimentin rabbit monoclonal antibody (1:1000) (Cell Signaling Technology, MA, USA), RBX1 rabbit monoclonal antibody (1:000) (Cell Signaling Technology, MA, USA) or CRKL mouse monoclonal antibody (1:1000) (Cell Signaling Technology, MA, USA). GAPDH rabbit monoclonal antibody (1:10000) (Cell Signaling Technology, MA, USA) was used as endogenous control. Protein expression was detected with WesternBright Quantum HRP substrate (Advansta, CA, USA), visualized on FUSION FX7 Image and Analytics System (Vilber Lourmat, Eberhardzell, Germany), and quantified using ImageJ v1.49.