Phospho β cateninser675

TM was purchased from Cell Signaling (Danvers, MA, USA). Matrigel and collagen type I were purchased from Corning (Bedford, MA, USA). Human TGF‐beta1 Quantikine ELISA Kit was purchased from R&D Systems (Minneapolis, MN, USA). DAPI and pyrvinium were purchased from Sigma (St. Louis, MO, USA). Phalloidin was purchased from Yuheng Biotechnology (Suzhou, China). Glycogen Periodic Acid Schiff stain kit was purchased from Solarbio Life Sciences. Puromycin was purchased from BioFroxx (Guangzhou, China). X‐tremeGENE transfection reagent was purchased from Roche (Mannheim, Germany). Antibodies used were as follows: human CD44–FITC and CD24–phycoerythrin and their respective isotype controls were obtained from BD Biosciences (Franklin Lakes, NJ, USA). β‐Catenin was purchased from Santa Cruz (Dallas, TX, USA). VE‐cadherin, integrin β1, BiP, CHOP, Smad2/3, Phospho‐Smad2, Phospho‐β‐Catenin (Ser33/37/Thr41) and Phospho‐β‐Catenin (Ser675) were purchased from Cell Signaling. Bmi‐1 was purchased from Epitomics (Burlingame, CA, USA). Short hairpin RNA (shRNA) encoding β‐Catenin and scramble control shRNA were purchased from TsingKe Biological Technology (Chengdu, China).

Overnight incubation was performed at 4 ℃ with rabbit primary antibodies against BIM (#2933, 1:1,000; Cell Signaling Technology) and PUMA (#4976; 1:1,000; Cell Signaling Technology). After washing with Tris-buffered saline and 0.1% polysorbate 20, membranes were incubated with horseradish peroxidase–conjugated secondary anti-rabbit IgG antibodies (#7074; 1:1,000; Cell Signaling Technology) at room temp for 1 h. 1 h incubation was performed at RT with rabbit primary antibodies against β-catenin (#8480, 1:1,000; Cell Signaling Technology), Phospho-β-Catenin (Ser675) (#4176, 1:1,000; Cell Signaling Technology), Wnt5a (ab235966, 1:1,000; Abcam). Regarding Klotho-GFP, 1 h incubation was performed at RT with Rat primary antibodies against Klotho (KO603, 1:1,000; Medicinal Chemistry Pharmaceutical Co.) and horseradish peroxidase–conjugated secondary anti-Rat IgG antibodies (5220–0365; 1:20,000; SeraCare). Immunoreactive protein was detected with enhanced chemiluminescence substrate, and band intensities were quantified by ImageQuant TL ver.8.1 (Cytiva). After visualization of the target protein, membranes were stripped and reincubated with antibodies against β-actin (#4967; Cell Signaling Technology)^{11 (link)}.