Total lrp6

Cells were lysed in RIPA buffer, proteins were separated on a 10% SDS-polyacrylamide gel by electrophoresis and transferred onto Nitran membrane (Sigma-Aldrich, USA). These were blocked with 5% non-fat dry milk and incubated with primary antibody (ZNF471 (1:3000; Sigma, USA), p-AKT (Ser473), total AKT, anti-CTNNB1, non-phospho-CTNNB1, CDH1, p-ELK-1(Ser383), total ELK-1, SNAI2, CCND1, c-MYC, DVL2, WNT3A, p-LRP6, total LRP6, β-actin (1:3000, Cell Signaling Technologies, USA), RAF1, (1:3000, Santa Cruz Technologies, USA) and SNAII, CDH2, VIM (1:3000, Cloud Clone, USA), Ki67, and Caspase 3 (1:3000, Novus Biological, USA)) overnight at 4°C and subsequently by secondary antibodies either anti-mouse IgG-HRP or anti-rabbit IgG-HRP (1:5000, Cell Signaling, USA). Proteins were visualized by SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific, USA), and imaging was performed with Image Quant LAS 4000 (GE Healthcare, USA).