Ecl detection agent

Proteins were separated on 10% Bis-Tris gels and transferred to Immobilon-P membranes (Millipore). PCNA antibodies (1:1000) were from Abcam. Antigen-antibody complexes were visualised by ECL detection agent (GE Healthcare).

Cells and tissue were first lysed with RIPA buffer (Beyotime Biotechnology, Shanghai, China) at room temperature for 1 h. The bicinchoninic acid method was used to measure the level of proteins. Proteins in the lysate were separated by electrophoresis and then transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% skimmed milk powder (Sigma-Aldrich) and then incubated with primary antibodies overnight at 4 °C. The following antibodies were used: anti-MEX3C (Cell signaling, #50,844, 1:1,000), anti-RUNX3 (Cell signaling, #9647, 1:1,500), anti-Suv39H1 (Novus, NBP1-21,367, 1:1,000), anti-E-cadherin (Abcam, ab231303, 1:1,500), anti-N-cadherin (Abcam, ab76011, 1:1,000), anti-Bcl-2 (Abcam, ab182858, 1:1,000), anti-Bax (Abcam, ab32503, 1:1,000), anti-Cleaved-caspase-3 (Cell signaling, #9661, 1:1,000), and anti-GAPDH (Abcam, ab8254, 1:2,000). The membranes were then washed in TBST and incubated with horseradish peroxidase-labeled secondary antibody. After washing in TBST again, protein bands were visualized using an ECL detection agent (GE Healthcare, Chicago, IL, USA) and ImageJ software. GAPDH were used as internal loading controls.