Protein Immunoblotting Assay Protocol

The protocol was operated in accordance with the descriptions in a previous paper [32 (link)]. Briefly, the segregated proteins were blotted on the membranes (Polyvinylidene Fluoride; Millipore, Bedford, MA, USA). Then, the membranes were incubated with unique primary antibodies overnight at 4°C after blocking for 1 hour. On the next day, the corresponding secondary antibody was used for the combination with the primary antibody, and the combined signals were appeared via adding the reagents of an enhanced chemiluminescence kit (Millipore). The primary antibodies against c-Myc (ab168727, 1:1000, Abcam, Cambridge, UK), Matrix metallopeptidase 9, (MMP-9, ab137867, 1:1000, Abcam), Ceaved-caspase-3 (ab49822, 1:1000, Abcam), Wnt5a (ab174963, 1:1000, Abcam), β-catenin (ab16051, 1:1000, Abcam), β-actin (ab179467, 1:2500, Abcam), and goat anti-rabbit secondary antibodies (ab205718, 1:5000, Abcam) were used in this study.