Anti wnt1

Total protein was isolated by radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, Inc.) and phenylmethanesulfonyl fluoride (PMSF). Protein lysates isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were then transferred to polyvinylidene fluoride (PVDF) membranes (Roche). Then the membrane was immunostained at 4°C by primary antibodies overnight. Primary rabbit antibodies used in the current study included anti-wnt1 [Cell Signaling Technology, Inc. (CST)]. Rabbit anti-GAPDH [Cell Signaling Technology, Inc. (CST)] was the loading control. Protein relative expression level was determined by Image Lab software.

RIPA buffer (Solarbio, Beijing, China) was used to isolate total proteins in cells and proteins were quantified by a NanoDrop 3000 (Thermo Fisher Scientific). Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) was used to separate proteins, and proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes. After that, membranes were blocked in skim milk for two hours at 37°C and then incubated with primary antibodies at 4°C overnight. Following two hours incubation with secondary antibody marked with horseradish peroxidase (HRP) (1:2000; Cell Signaling Technology, Danvers, MA, USA). Chemiluminescence was performed using an ECL detection kit (Beyotime, Shanghai, China). The primary antibodies were as follows: anti‐N‐cadherin (1:1500; Cell Signaling Technology, Danvers, MA, USA), anti‐E‐cadherin (1:1000; Cell Signaling Technology), anti‐Vimentin (1:1000; Cell Signaling Technology), anti‐Snail (1:1000; Cell Signaling Technology), anti‐MCL‐1 (1:1000; Cell Signaling Technology), anti‐Wnt1 (1:1000; Cell Signaling Technology), anti‐C‐myc (1:1000; Cell Signaling Technology), anti‐CyclinD1 (1:1000; Cell Signaling Technology), anti‐β‐catenin (1:1000; Cell Signaling Technology) and anti‐GAPDH (1:4000; Cell Signaling Technology).

The proximal and distal portions of the colon were freshly isolated and embedded in paraffin wax after fixation with 10% neutral buffered formalin. Immunostaining was performed on paraffin-embedded sections (4 μm) of mouse colons and human colon tissue. Slides were incubated in 3% hydrogen peroxide for 20 minutes at room temperature to block endogenous peroxidase activity and then in 5% BSA in PBS for 30 minutes to reduce nonspecific background. The permeabilized tissue samples were incubated with anti-Wnt1 (1:100, Cell Signaling) and anti–β-catenin (1: 100; BD, San Jose, CA) for 10 to 12 hours at 4°C. Samples were then incubated with DAPI for 10 minutes at room temperature. Tissues were mounted with SlowFade (SlowFade AntiFade Kit, Molecular Probes) and then cover slipped. The edges were sealed to prevent drying. Specimens were examined with a Leica SP5 scanning confocal microscope.