Protein Expression and Localization Analysis

Briefly, proteins were prepared by radioimmunoprecipitation assay (RIPA) buffer and quantified by a BCA Protein Assay Kit (Beyotime, China). The nuclear protein was obtained using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) following the manufacturer’s protocol. Samples were subjected to 10% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked and then sequentially incubated with the primary antibody, the secondary antibody, and detected by chemiluminescence. The antibodies used included primary antibodies against RUNX3, E-cadherin, Vimentin, N-cadherin, β-catenin, c-Myc, CD44, CCND1, GAPDH (glyceraldehyde-3-phosphate dehydrogenase, as negative control), and β-actin (Proteintech, USA). IF assays were conducted as reported previously.¹² (link) In brief, cells grown on slides, including the transfected GC cells, were successively incubated with primary antibodies (against E-cadherin and Vimentin) and fluorescent secondary antibodies (Beyotime) and counterstained with 4,6-diamidino-2-phenylindole (DAPI) (nucleus). Cells on the slides were observed and imaged using a ZEISS LSM800 fluorescence microscope (Carl Zeiss AG, Germany).