Rabbit anti human β catenin

Whole constructs were fixated in paraformaldehyde (4%), for 20 min, and then permeabilized with 0.3% Triton X-100 (Bio Lab Ltd.), for 10 min. Constructs were then washed with PBS and immersed overnight in BSA solution (5%; Millipore). Samples were then incubated with goat antihuman VE-cadherin (1:100; Santa Cruz), mouse antihuman Yes-associated protein (YAP) (1:100; Santa Cruz), mouse antihuman NG2 (1:100; Santa Cruz), rabbit antihuman vWF (1:150; Abcam), rabbit antihuman β-catenin (1:100; Sigma-Aldrich), or mouse antihuman Ki67 (1:20, DAKO) antibodies, overnight, at 4°C. Constructs were then treated with Cy3-labeled (1:100; Jackson Immunoresearch Laboratory), Cy5-labeled (1:100; Jackson Immunoresearch Laboratory), or Alexa-488 (1:400; ThermoFisher Scientific) antibodies, mixed with DAPI (1:1000; Sigma-Aldrich), for 2 h, at room temperature. For the phalloidin staining, constructs were treated with FITC phalloidin (1:100; Sigma-Aldrich) and DAPI for 20 min. For the mitomycin experiment, mitomycin-treated cells and control cells without mitomycin were fixated in paraformaldehyde (4%), for 20 min on day 10 of culture, and then incubated in a 30% (wt/vol) sucrose solution overnight, embedded in optimal cutting temperature compound (Tissue-Tek) and frozen for subsequent cryosectioning (5–20 µm). Standard protocols were used for H&E and Masson’s trichrome staining of the sections.

Cultured CRC cells were lysed in RIPA Lysis Buffer (Beyotime) supplemented with a protease inhibitor and phosphatase inhibitor (Roche). Protein lysates were separated by SDS‒PAGE and transferred onto nitrocellulose membranes (Millipore). The membranes were sequentially blocked with 10% skim milk in TBST (150 mM NaCl, 120 mM Tris–HCl, pH of 7.4, and 0.05% Tween 20), incubated with primary antibodies, and incubated with secondary HRP-conjugated antibodies (Cell Signaling Technology). The antibodies used were as follows: rabbit anti-human DKK4 (Abcam), mouse anti-human β-actin (Sigma‒Aldrich), rabbit anti-human β-catenin, rabbit anti-human nonphospho (active)-β-catenin (Ser33/37/Thr41), rabbit anti-human phospho-β-catenin (Ser552), rabbit anti-human AKT1, AKT2, phospho-AKT2, FZD6, JUN, LRP5, LRP6, MMP2, and MMP3 (Cell Signaling Technology), and goat anti-rabbit or goat anti-mouse IgG (ZSGB). β-actin was used as an internal control. The immunoreactive protein complexes were detected with enhanced chemiluminescence reagents (ZETA) and scanned by the Molecular Imager ChemiDox XRS+ Imaging System with Image Lab software (Bio-Rad).