Precast gel

RIPA buffer mixed with protease inhibitor (Thermo Fisher Scientific, USA) was used to lyse cells for harvest of total protein. The protein was separated by 4–20% Precast-Gel (Solarbio, China) and transferred to a PVDF membrane (Millipore, USA). Then 5% bovine serum albumin was used to block the membrane for 1 h at room temperature, and incubated with primary antibody overnight at 4℃. The following primary antibodies were used: anti-Flag (Origene, USA), anti-SATB2 (Abcam, USA), anti-β-catenin, anti-active β-catenin (Cell Signaling Technology, USA), anti-β-actin (Zhongshanjinqiao, China), anti-RUNX2 (Biorbyt, England), anti-JHDM1D (Abcam, USA), anti-DKK1 (Santa Cruz, USA), anti-H3K9me2, anti-H3K27me2 (Abcam, USA). HRP-conjugated secondary antibody (1:10,000; Zhongshanjinqiao, China) was then used to incubate the membrane for 1 h at room temperature. At last, we used ECL and Super Signal detection reagents (Thermo Fisher Scientific, USA) to detect the membrane.

Protein samples were collected using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma) for 30 min on ice and quantified using a bicinchoninic acid protein assay kit (KeyGen, China). Equivalent amounts of cell lysate were separated using a precast gel (Solarbio, China) and transferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes were then blocked for 1 h at room temperature and immunoblotted at 4°C with primary antibodies overnight. The next day, membranes were washed with Tris‐Buffered Saline and Tween (TBST) at least 3 times and then incubated with appropriate secondary antibodies (Abmart, China). Bands were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA). The antibodies used are listed in Table S7.