Ha 3724

Skin and tumors were fixed in 10% buffered formalin and embedded in paraffin. Sections were stained with H&E, and histopathological evaluation was performed by an experimented observer veterinarian expert in animal pathology (R.A.G.F.). Immunostaining was performed using antibodies against HA (3724, Cell Signaling Technology), K5 (PRB-160P), K10 (PRB-159P), and filaggrin (PRB-417P) (Biolegend, San Diego, CA, USA); K13 (AE8; Abcam, Cambridge, UK); PECAM/CD31 SC-1506 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); CYLD (SAB4200061) and sma (C-6198) (Sigma-Aldrich, St Louis, MO, USA); and BrdU (11170376001; Roche, Mannheim, Germany). Sections were incubated with a biotinylated secondary antibody and then with streptavidin conjugated to horseradish peroxidase (DAKO A/S, Glostrp, Denmark). Antibody localization was determined using 3,3-diaminobenzidine (DAB) (Vector Laboratories; Burlingame, CA, USA).

HA (#3724) and FLAG (#8146 and #14793) antibodies were purchased from Cell Signaling Technology. β-Actin (sc-4777) antibody was purchased from Santa Cruz Biotechnology. Protease inhibitor cocktail (P8340) and crystal violet (C0775) were purchased from Sigma. Nitro Blue tetrazolium chloride (J60230) was purchased from Alfa Aesar. MEM nonessential amino acids and ECL plus western blotting detection reagent were purchased from Thermo Fisher Scientific. Saponin (S0019-25G) was purchased from TCI America. Sep-Pak C18 cartridge was purchased from Waters.

Primary antibodies against Myc (sc-40), β-catenin (sc-7199), VBP1 (sc-390465), and pVHL (sc-135657) were purchased from Santa Cruz Biotechnology. Antibodies against TCF7L2 (2569), GFP (2555), and HA (3724) were obtained from Cell Signaling Technology. The antibody against TCF7L1/L2 (ab12065) was provided by Abcam. The antibody against GAPDH (D110016) was purchased from BBI Life Sciences (Crumlin, UK). The antibody against histone H3 (P30266) was obtained from Abmart. The FLAG M2 antibody (F1804) was provided by Sigma-Aldrich. The antibody against β-actin (abs137975) was obtained from Absin Bioscience. Small molecules CHX (66-81-9) and BIO (667463-62-9) were purchased from Sigma-Aldrich. MG132 (HY-13259) was from MedChemExpress.

Specific antibodies for Flag (M3165), α‐tubulin (T6199), and β‐actin (A2228) were acquired from Sigma (MO, USA). Those antibodies for Olfm4 (39141), β‐TrCP (4394), and HA (3724) were acquired from Cell Signaling Technology (MA, USA); those for Ki67 (ab15580), Lgr5 (ab75732), and lysosome (ab2408) were from Abcam (Cambridge, UK); those for β‐catenin (610153) were from BD Biosciences (San Jose, CA); those for SOX9 (AB5535) were from EMD Millipore (MA, USA); those for GSK3α/β (sc‐7291) and Laminin A/C (sc‐7292) were from Santa Cruz (Santa Cruz, CA); and those for MST4 and phospho‐β‐catenin (Thr40) were produced by Shanghai Immune Biotech (Shanghai, China). Secondary antibodies for Alexa Fluor 568 goat antirabbit IgG (H+L) (A11011), Alexa Fluor 647 donkey antigoat IgG (H+L) (A21447), and Alexa Fluor 488 goat antirabbit IgG (H+L) (A11001) were acquired from Invitrogen (Carlsbad, CA).