2.5 Immunoblot Analysis
For extraction of proteins, cells or mice-skin tissues were treated with RIPA buffer (20 mM Tris-HCl -pH 7.4, 150 mM NaCl, 1 % Triton X-100, 0.1 mM DTT, 1 mM EDTA, 1 mM EGTA, and 1 mM PMSF) containing the protease and phosphatase inhibitor cocktail (Topscience) trailed by sonication. The protein extraction was then fractionated by SDS‐PAGE and passed-on to PVDF membranes (Cat# ISEQ00005, Millipore, Burlington, MA, USA). Western blotting was accomplished with the subsequent primary antibodies: anti‐β‐catenin (1:2,000, Santa Cruz Biotechnology Cat# sc‐7963, RRID:AB_626807), anti‐LGR5 (1:1,000, Abcam Cat# ab75732, RRID:AB_1310281), anti‐SOX2 (1:1,000, Cell Signaling Technology Cat# 23064, RRID:AB_2714146), anti‐Flag (1:5,000, Sigma‐Aldrich Cat# F1804, RRID:AB_262044), anti‐GAPDH (1:5,000, Transgen Biotech Cat# HC301–02, RRID:AB_2629434), anti-LRP6 antibody (1:1000 Cell Signaling Technology Cat# 2560, RRID:AB_2139329), anti-CK1ε antibody (1:1000 Cell Signaling Technology Cat# 12448, RRID:AB_2797919), anti-active β-catenin antibody (1:1000 Cell Signaling Technology Cat#8814, RRID:AB_11127203), anti-LEF1 antibody (1:1000 Cell Signaling Technology Cat#76010, RRID:AB_2799877), anti-Axin2 antibody (1:1000 Cell Signaling Technology Cat# 2151, RRID:AB_2062432), anti-V5 antibody (1:1000 Cell Signaling Technology Cat#13202, RRID:AB_2687461), anti-Survivin antibody (1:1000 Santa Cruz Biotechnology Cat# sc-17779, RRID:AB_628302), anti-pLRP6 antibody (1:1000 Cell Signaling Technology Cat# 2568 RRID:AB_2139327), anti-GSK-3-beta (1:1000 Cell Signaling Technology Cat#9315S RRID:AB_490890), anti-Twist1 (1:1000 Cell Signaling Technology Cat#46702S RRID:AB_2799308), anti-Phospho-GSK-3β (1:1000 Cell Signaling Technology Cat#9322S RRID:AB_2115196), and Anti cytokeratin 17 (1:1000, Abcam Cat# ab53707, RRID:AB_869865). Western blotting was carried out with the chosen primary antibodies with nurture of 4 °C overnight. Next, the PVDF membranes were mounting with HRP (Horseradish Peroxidase) conjugated goat anti‐rabbit (1:10,000, Thermo Fisher Scientific Cat# A16096, RRID:AB_2534770) or anti-mouse (1:10,000, Thermo Fisher Scientific Cat# A16066, RRID:AB_2534739) or IgG at room temperature for 1 h. After incubating with substrate of ECL Plus-Western-Blotting (Thermo Fisher Scientific, Cat# 32132), the immunoblots were established by Chemiluminescent Imaging System (Tanon 5200, Shanghai, China).