Bm0627

Primary antibodies against Snail1 (cat. no. 125918) were purchased from GeneTex, Inc. (Irvine, CA, USA). Primary antibodies against Wnt1 (ab85060), Wnt8a (ab130930), phosphorylated glycogen synthase kinase 3β (P-GSK3β; ab130937) and GAPDH (ab37168) were obtained from Abcam (Cambridge, UK). Primary antibodies against Wnt3a (bs1700R) and Wnt5a (bs1948R) were purchased from BIOSS (Beijing, China). The primary antibody against Wnt11 (sc50360) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibodies against β-catenin (BA0426) and β-actin (BM0627) were obtained from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). The antibody against GSK3β (22104-1-AP) was purchased from Wuhan Sanying Biotechnology (Wuhan, China). The bicinchoninic acid (BCA) protein assay kit was purchased from Beyotime Institute of Biotechnology (Haimen, China). TRIzol (252250AX) was obtained from Aidlab Biotechnologies Co., Ltd. (Beijing, China). M-MLV Reverse Transcriptase (RNase H; CO2010A) and ddH₂O (DNase/RNase Free; C1D230A) were purchased from GeneCopoeia, Inc. (Rockville, MD, USA) and RNase Inhibitor (I21222) was obtained from TransGen Biotech, Inc. (Beijing, China).

To measure the protein expression of phosphorylaed GSK-3β at Tyr-216 (p-Tyr216 GSK-3β) and β-catenin, we conducted western blot analysis. Total proteins were separated on 10% SDS-PAGE gels and then transferred onto polyvinylidene fluoride blotting membranes. After blocking non-specific binding with 5% BSA in Tween-Tris-buffered saline, the membranes were incubated with the following primary antibodies: anti-p-Tyr216 GSK-3β (ab75745; 1:500 dilution; Abcam, Cambridge, CA, USA), anti-β-catenin (ab32572; 1:1,000 dilution; Abcam) and anti-β-actin (BM0627; 1:200 dilution; Wuhan Boster Biological Technology, Ltd.) antibodies overnight. The membranes were then incubated for 2 h with secondary antibodies (ZDR-5306, 1:2,000 dilution; Zhongshan Golden Bridge Biotechnology, Co., Ltd., Beijing, China) labeled with horseradish peroxidase. The immunoreactive proteins on the blots were visualized using ECL™ western blot detection reagents, and the signals were detected using Image Station 4000R (Kodak, Rochester, NY, USA).