Horseradish peroxidase conjugated

Cells were lysed in RIPA buffer combined with proteasome inhibitor and phosphatase inhibitors (Beyotime). Equal amounts of proteins were separated by 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, then separated target proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Shanghai, China). After blocking in 5% non-fat milk for 60 min, the membranes were incubated overnight at 4 °C with antibodies specific to GAPDH (1:2000; Abcam, Shanghai, China), APELIN (1:2000; Abcam, Shanghai, China), RUNX2 (1:1600; Abcam, Shanghai, China), COL1A1 (1:1000; Abcam, Shanghai, China), non-phosphorylated (active) β-catenin (1:1000; Abcam, Shanghai, China), or total β-catenin (1:1000; Abcam, Shanghai, China). After washing with TBST three times (10 min each), the membranes were incubated with secondary antibodies (horseradish peroxidase-conjugated, Beyotime) for 1 h at room temperature. After washing three times with TBST, proteins were detected using enhanced chemiluminescence blotting reagents (Millipore). Signal intensity was measured by Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA, USA).

For detailed procedure, refer to the previous study 10. Briefly, cells were lysed using RIPA protein extraction reagent (Beyotime, Shanghai, China) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentration was measured using the Bio‐Rad protein assay kit (Bio‐Rad, Beijing, China). Approximately 30 μg of protein extract was separated by 10% SDS/PAGE, then transferred to nitrocellulose membrane (Sigma‐Aldrich, St Louis, MO, USA) and incubated with the primary antibodies against E‐cadherin (cat. no. 20874‐1‐AP), Slug (cat. no. 12129‐1‐AP), Nanog (cat. no. 14295‐1‐AP), aldehyde dehydrogenase 1 (ALDH1) A1 (cat. no. 15910‐1‐AP), Dicer (cat. no. 20567‐1‐AP) and β‐actin (cat. no. 60008‐1‐Ig), which were purchased from Proteintech. The secondary antibodies (cat. no. A0216 and cat. no. A0239), horseradish peroxidase‐conjugated, were purchased from Beyotime. An Enhanced Chemiluminescence Plus kit (cat. no. YP0071; Yi Fei Xue Biotechnology, Nanjing, China) was used to develop an image in a Tanon 5200 machine (Tanon, Shanghai, China).