Anti β catenin

Cells were harvested and lysed in RIPA buffer (Beyotime, #P0013E) at 48 h after siRNA transfection. Protein concentration was detected using a BCA kit (Beyotime, #P0012S). The extracted protein was separated in a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. The membranes were blocked in TBST (Tris-buffered saline, 0.1% Tween 20) buffer containing 5% silk milk powder for 2 h at room temperature, then cut according to the molecular weight of the target proteins, and incubated with primary antibodies at 4 °C overnight. The membranes were washed three times followed by incubation with secondary antibody for 1 h at room temperature. The ECL system and Image Lab software were used to detect signals and analyze protein quantitatively, respectively. The primary antibodies used were as follows: anti-LRRFIP1 (Santa Cruz Biotechnology, #sc-101168, 1:500 dilution), anti-p-AKT (Cell Signaling Technology, #4060, 1:1000 dilution), AKT (Cell Signaling Technology, #4691, 1:1000 dilution), anti-p-GSK-3β (Beyotime, #AG753, 1:1000 dilution), anti-GSK-3β (Beyotime, #AG751, 1:1000 dilution), anti-β-catenin (Beyotime, #AF0069, 1:2000 dilution), and anti-GAPDH (ZS-GB Biotech, #TA-08, 1:5000 dilution).

All plasmids were purchased from Genomeditech (Shanghai, China). pGLMV-GCM1 was subcloned into a pGMLV-6395 expression vector. pNanog-HA was subcloned into a pcDNA3.1 expression vector. The primary antibodies used were: anti-GCM1 (#sc-101173, 1:50; Santa Cruz, USA); anti-β-catenin (#D1018, 1:1000; CST, USA); anti-Nanog (ab214549,1:1000; Abcam, USA); anti-β-catenin (#AF0069, 1:1000; Beyotime, China); anti-TCF4 (sc-166699; 1:500, Santa Cruz, USA); anti-Axin2 (#21515, 1:2000; CST, USA); anti-histone H3 (#ab21054, 1:5000; Abcam, USA); and anti-Atoh1 (#ab168374, 1:1000, Abcam, USA).