Nitran membrane

Whole-cell protein (20–50 μg) was initially separated using 8% SDS-PAGE and subsequently, transferred onto Nitran membrane (Sigma-Aldrich, USA). Next, the membranes were blocked with BSA (5%), and incubated separately with anti-DOC2B (1:5000; Proteintech, USA), anti-CTNNB1 and CDH1 (1:3000) (Developmental Studies Hybridoma Bank, University of Iowa, USA), p-ELK-1(Ser383), total ELK-1, p-AKT (Ser473), total AKT, p-ERK1/2(Thr202/Tyr204), total- ERK1/2 (Thr202/Tyr204), p-p38MAPK (Thr180/Tyr 182), total- p38MAPK, MacroH2A1.2, Tri methyl H3 lys9, SNAI1, SNAI2, ZEB1 and β-actin (1:3000, Cell Signaling Technologies, USA), CCNE (HE12), GSK3α/β, CDKN2A, CDKN1A, CDKN1B (1:3000, Santa Cruz Technologies, USA) TWIST2 (1:2000, Abcam, Cambridge, USA) and CLDN1, CDH2 VIM (1:3000, Cloud Clone, USA) at 4 °C, and then with anti-mouse IgG-HRP or anti-rabbit IgG-HRP (1:5000) (Cell Signaling, USA) secondary antibodies. SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific, USA) was used for visualization of proteins in the membranes using Image Quant LAS 4000 (GE Healthcare, USA).

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).