Anti dkk1

Cell lysates were measured for protein concentration using BCA kit (Beyotime Biotechnology, Shanghai.). 50 μg of lysates was separated by 10% SDS/PAGE. The proteins were transferred to nitrocellulose membrane. After being blocked in 5% milk (w/v) at room temperature for 1 hour, the membranes were incubated at 4 °C overnight with primary antibodies (1:1,000). Following 1×PBST washing, the membranes were incubated with secondary antibodies (1:3,000) at room temperature for 1 hour followed by ECL staining. The following antibodies were used: anti-Snail1 (sc-271977, Santa Cruz Biotechnology), anti-Slug (sc-166902, Santa Cruz Biotechnology), anti-ZEB1 (sc-515797, Santa Cruz Biotechnology ), anti-ZEB2 (sc-271984, Santa Cruz Biotechnology), anti-Twist (sc-81417, Santa Cruz Biotechnology), anti-DKK1 (sc-374574, Santa Cruz Biotechnology), anti-BMI-1 (sc-390443, Santa Cruz Biotechnology), anti-Oct4 (2750S, Cell Signaling Technology), anti-Nanog (4903S, Cell Signaling Technology), anti-Vimentin (sc-32322, Santa Cruz Biotechnology), anti-GAPDH (5174, Cell Signaling Technology), and anti-β-actin (sc-47778, Santa Cruz Biotechnology). HRP-conjugated anti-rabbit IgG (7074S, Cell Signaling Technology) and HRP- conjugated anti-mouse IgG (7076S, Cell Signaling Technology) were used as secondary antibodies.

Cells were lysed using RIPA or SDS lysis buffer supplemented with protease inhibitors (Complete Mini, Sigma-Aldrich). The following primary antibodies were used: anti-EHMT2 (#3306S, 1:300, Cell Signaling), anti-MHC (#sc-32732, 1:300, Santa Cruz Biotechnology), anti-Myogenin (#sc-12732, 1:250, Santa Cruz Biotechnology), anti-DKK1 (#sc374574, 1:300, Santa Cruz Biotechnology), anti-active-ß-catenin (#05–665, 1:500, Merck Millipore), anti-H3K9me2 (#9753S, 1:1000, Cell Signaling), anti-ß-actin (#A2228, 1:10,000; Sigma-Aldrich), and anti-H3 (#ab1791, 1:10,000; Abcam). Appropriate secondary antibodies (IgG-Fc Specific-Peroxidase) of mouse or rabbit origin (Sigma Aldrich) were used.

For western blotting assay, intestinal epithelial tissues were lysed in lysis buffer (Beyotime, China) with 1% PMSF (Phenylmethylsulfonyl fluoride). After quantification using a BCA protein assay kit (Beyotime, China), 30 μg of total protein was separated by 10% SDS-PAGE under denaturing conditions and transferred to PVDF membranes (GE Healthcare). Membranes were blocked in 5% nonfat dry milk in incubation buffer and incubated with primary antibodies, followed by incubation with the secondary antibody and chemiluminescent detection system (Pierce). The primary antibodies were: anti-GAPDH (Sigma), anti-β-Tubulin (Sigma), anti-CyclinD1 (Santa Cruz), anti-c-Myc (Santa Cruz), anti-β-Catenin (Sigma), anti-Dkk1 (Santa Cruz), anti-Gsk3β (Abcam), anti-Axin1 (Cell Signaling), anti-p-Smad2/3 (Cell Signaling), anti-p21(Santa Cruz), anti-Smad4 (Santa Cruz), anti-p-Smad1/5/8 (Cell Signaling), anti-Bmpr1a (Abcam), anti-p65 (Cell Signaling), anti-STAT3 (Cell Signaling), anti-p-p65 (Cell Signaling), anti-p-STAT3 (Cell Signaling).

Intestines were rinsed with 1x DPBS, fixed in 10% formalin, paraffin-embedded and sectioned at 5 μm. Sections were stained with hematoxylin and eosin (H&E). For immunohistochemistry, antigen retrieval was performed by heating slides in 0.01 M citrate buffer (pH 6.0) in a microwave. Sections were then immunostained using ABC peroxidase method (Vector labs) with diaminobenzidine (DAB) as the enzyme substrate and hematoxylin as the counterstain. For immunofluorescence staining, paraffin sections were microwave pretreated in 0.01 M citrate buffer (pH 6.0), and incubated with primary antibodies, then incubated with secondary antibodies (Invitrogen) and counterstained with DAPI in the mounting medium (Vector labs). The following antibodies were used: anti-Ki67 (1:150, Leica), anti-GFP (1:200, Abcam), anti-Axin1 (1:100, Cell Signaling), anti-Gsk3β (1:2000, Abcam), anti-Dkk1 (1:50, Santa Cruz), anti-β-Catenin (1:500, Sigma), anti-BrdU (1:50, Abcam), anti-cleaved Caspase3 (1:100, Cell Signaling), anti-p-Smad1/5/8 (1:200, Cell Signaling), anti-p-Smad2/3 (1:200, Cell Signaling), anti-CyclinD1 (1:50, Abcam), anti-p65 (1:400, Cell Signaling), anti-p-STAT3 (1:800, Cell Signaling).

Western blot analysis and immunofluorescence were preformed as previously described⁴; the primary antibodies are listed in Table S1, including anti‐GAPDH (1:10 000, rabbit monoclonal, Abcam), anti‐PCNA (1:1000, mouse monoclonal, Abcam), anti‐Ki67 (1:1000, mouse monoclonal, Abcam), anti‐cyclin E1 (1:1000, rabbit monoclonal, Abcam), anti‐cyclin D1 (1:10 000, rabbit monoclonal, Abcam), anti‐cyclin A2 (1:2000, rabbit monoclonal, Abcam), anti‐cyclin B1 (1:2000, rabbit monoclonal, Abcam), anti‐cleaved‐PARP (1:10 000, rabbit monoclonal, Abcam), anti‐cleaved‐caspase‐3 (1:1000, rabbit polyclonal, CST), anti‐IGFBP3 (1:1000, rabbit monoclonal, CST), anti‐DKK‐1(1:1000, mouse monoclonal, Santa Cruz), anti‐DKK‐3 (1:1000, rabbit monoclonal, Abcam), anti‐P‐Gsk3β (1:1000, rabbit polyclonal, Abcam), anti‐Gsk3β (1:1000, mouse monoclonal, Abcam), anti‐β‐catenin (1:5000, rabbit monoclonal, Abcam), anti‐AKT (1:1000, mouse monoclonal, Abcam), anti‐P‐AKT (1:1000, mouse monoclonal, Abcam), anti‐PI3K (1:1000, rabbit polyclonal, Abcam), P‐PI3K (1:1000, rabbit polyclonal, CST), anti‐IGF‐1R (1:1000, rabbit monoclonal, CST), anti‐P‐IGF‐1R (1:1000, rabbit monoclonal, CST), anti‐N‐cadherin (1:5000, rabbit monoclonal, Abcam), anti‐Bcl‐2 (1:1000, mouse monoclonal, Abcam) and anti‐Bax (1:1000, mouse monoclonal, Abcam).