Dact2

Cells were collected 48h after transfection and cell lysates were prepared using ice-cold Tris buffer (20 mmol/L Tris; pH 7.5) containing 137 mmol/L NaCl, 2 mmol/L EDTA, 1% Triton X, 10% glycerol, 50 mmol/L NaF, 1 mmol/L DTT, PMSF, and a protein phosphatases inhibitor (Applygen Tech. Beijing, China). Antibodies were diluted according to manufacturer’s instructions. Primary antibodies are as follows: DACT2 (OriGene Tech. MD, USA), active-β-catenin (Millipore, CA, USA), p-β-catenin (Bioworld Technology, MN, USA), total-β-catenin (Cell Signaling Tech, MA, USA), myc (Proteintech, IL, USA), cyclin D1 (Proteintech, IL, USA), cyclin E1 (Proteintech, IL, USA) and actin (Bioworld Technology, MN, USA).

Protein samples from unexpressed and stably re-expressed KYSE150 and KYSE450 cells were collected and western blots were performed as described previously [44 (link)]. Antibodies were diluted according to the manufacturer's instructions. Primary antibodies included DACT2 (Cat: TA306668, OriGene Tech, MD, USA), c-Myc (Cat: 10828-1-AP, proteintech, IL, USA), cyclin B1 (Cat: 55004-1-AP, proteintech, IL, USA), CDC2 (Cat: 19532-1-AP, proteintech, IL, USA), p-CDC2 (Y15) (Cat: #9111, Cell Signaling Tech, MA, USA), MMP2 (Cat: BS1236, Bioworld Tech, MN, USA), MMP9 (Cat: BS1241, Bioworld Tech, MN, USA), Anti-Active-β-Catenin, clone8E7 (Cat: #05-665, Millipore, CA, USA), p-β-catenin (S37) (Cat: BS4739, Bioworld Tech, MN, USA), β-catenin (D10A8) XP^® (Cat: #8480, Cell Signaling Tech, MA, USA) and β-actin (Cat: AF0003, Beyotime Biotech, Jiangsu, China).

Western blotting was performed as previously described [31 (link)]. Aliquots of 40 μg of protein lysate were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Membranes were incubated with DACT2 (TA306668, Origene), active β-catenin (#4270; Cell Signaling Technology), total β-catenin (#9562; Cell Signaling Technology), MMP9 (ab76003, Abcam), MMP2 (ab86607, Abcam), c-Myc (#13987, Cell Signaling Technology), Cyclin D1(sc-450), p-GSK3β (sc-373800), Cdc25c (sc-13138), Cdc2 (sc-54), p-Cdc2 (pY15.44) (sc-136014), β-actin (sc-8432) (all from Santa Cruz Biotechnology, CA, USA), or CyclinB1 (ab32053, Abcam) primary antibodies. Proteins were visualized using an Immobilon Western Chemiluminescent HRP Substrate kit (Millipore Corporation, Billerica, MA, USA).