Total β catenin

The following antibodies were used in Western blotting and immunofluorescence:
phospho-GSK3α/β (CST 8566), phospho-β-Catenin (Ser33/37/Thr41) (CST9561), non-phospho (Active) β-Catenin (Ser33/37/Thr41) (CST 8814), total β-Catenin (CST 9587), phospho-cyclin D1 (CST 3300), PPARγ (CST 2443), p27 (CST 2552), Ki-67 (CST 9129), phospho-c-Jun (CST 9164), c-Myc (CST 13987), Cytochrome c (CST 4280),GFAP (CST 3670), vimentin (CST 5741), NF-kB (CST 8242), c-Fos (CST 2250) from Cell Signaling Technology (Danvers, MA, US); Lamin B1 (10H34L18), Cyclin D1 (MA5-14512), Bcl-2 (13-8800), phospho-CREB (MA1-083), Ki-67 (MA5-14520) from Invitrogen, Thermo Fisher Scientific, and Phospho-JNK (sc-6254) from Santa Cruz Biotechnology (Dallas, TX, USA).

Colon cancer cells or tumor organoids were collected and detergent-solubilized cell lysates were obtained as described previously^{8 (link),14 (link)}. The NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo) was used to separate cytoplasmic and nuclear fractions. Equal amounts of total cell lysates were resolved by SDS–PAGE and subjected to Western blot analysis. The following antibodies, including CPT1A, acetylated-lysine (pan-Lys, #9441) acetyl-histone H3 (Lys9) (#9649), acetyl-histone H3 (Lys27) (#8173), histone H3 (#14269), active-β-catenin (#8814), total β-catenin (#8480), were purchased from Cell Signaling; the acetyl-α-tubulin (T7451) and β-actin (A1978) antibodies were from Sigma-Aldrich; and the total α-tubulin (sc-5286) and Lamin A/C (sc-20681) antibodies were from Santa Cruz.

PTEN (138G6) Rabbit Monoclonal antibody, Phospho-cdc2 (Tyr15) antibody, Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) Rabbit Monoclonal antibody, p44/42 MAPK (Erk1/2) Rabbit Monoclonal antibody, Phospho-β-Catenin (Ser33/37/Thr41) antibody, Total-β-Catenin antibody, Phospho-c-Myc (Ser62) Rabbit Monoclonal antibody, p53 Rabbit Monoclonal antibody, Non-phospho (Active) β-Catenin (Ser33/37/Thr41) Rabbit Monoclonal antibody were obtained from Cell Signaling Technology (CST). Mouse anti-human C-myc monoclonal antibody, anti-MMP-9 antibody were obtained from Millipore.

Ten-μg protein samples were run in 10% SDS-PAGE gel and transferred onto a polyvinylidene fluoride (PVDF) membrane using the Bio-Rad Mini-Protean system (Bio-Rad Laboratories, Inc, Hercules, CA, USA). Non-specific binding was blocked by incubating the membranes in 5% skimmed milk in tris-buffered saline with Tween 20 (TBST) for 1 h and then overnight at 4 °C with the antibodies against total β-catenin (1:1000, Cell Signaling Technology, Danvers, MA, USA), free β-catenin (1:1000, Cell Signaling Technology), p-GSK-3β (1:1000, Cell Signaling Technology), GSK-3β (1: 1000, Cell Signaling Technology), TCF1/TCF7 (1:1000, Cell Signaling Technology), LEF1 (1:1000, Cell Signaling Technology), p-Stat3 (1:1000, Santa Cruz, California, USA), Stat3 (1:1000, Santa Cruz), KLF4 (1:1000, Santa Cruz), c-Myc (1:1000, Santa Cruz), vimentin (1:1000, Cell Signaling Technology), Slug (1:1000, Cell Signaling Technology), and β-actin (1:500, Santa Cruz). After overnight probing with primary antibody, the membranes were incubated with horseradish peroxidise (HRP)-linked secondary antibodies for 1 h, then washed with PBS thrice. The protein band signals were detected and developed using an enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific Inc, Waltham, MA, USA). Protein bands were quantified using ImageJ software.

Total proteins from cell lines and tissue samples were extracted by ice‐cold Mammalian Protein Extraction reagent (Thermo Fisher Scientific, Inc.) containing 1% protease inhibitor cocktail (Merck KGaA, Darmstadt, Germany). Protein concentration was assessed by the bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, Inc.). Samples (20 μg protein/lane) were separated by electrophoresis on SDS‐PAGE gel (8 to 10%), followed by transferring onto the polyvinylidene difluoride (PVDF) membrane (Bio‐Rad Laboratories, Inc., Hercules, CA, USA). Thereafter, the PVDF membrane was subjected to blockage by Blocking One (Nacalai Tesque, Kyoto, Japan), incubated with primary antibodies against GAPDH (5174, 1:1000 dilution, Cell Signaling Technology), STK4 (14,946, 1:1000 dilution, Cell Signaling Technology), p‐β‐catenin (9566, 1:1000 dilution, Cell Signaling Technology), and total β‐catenin (8480, 1:1000 dilution, Cell Signaling Technology) at 4°C overnight and horseradish peroxidase (HRP)‐conjugated secondary antibodies (7074, 1:1000 dilution, Cell Signaling Technology) at room temperature for 1 h, successively. Protein bands were visualized by enhanced chemiluminescence (ECL, PerkinElmer, Inc.). Gray density was quantified by Quantity One software (version 4.62; Bio‐Rad Laboratories, Inc.). Relative protein expression was normalized to GAPDH.

Radioimmunoprecipitation assay (RIPA) buffer with a phosphatase inhibitor cocktail and a protease inhibitor (Sigma, St. Louis, MO, USA) was used to isolate proteins from cells. The protein concentration was measured using the bicinchoninic acid (BCA) assay (Thermo Scientific, Rockford, IL, USA). Standard Western blot techniques were performed as previously described [49 (link)]. The antibodies used were GAPDH (1:1000) (#2118, Cell Signaling Technology, Danvers, MA, USA), FLAG-tag (1:1000) (#14793, Cell Signaling Technology), FZD10 (1:500) (41686, Signalway Antibody, MD, USA), total β-catenin (1:1000) (#2677, Cell Signaling Technology), active β-catenin (1:1000) (#19807s, Cell Signaling Technology), CCND1 (1:1000) (cyclinD1, #1677-1, Epitomics), c-Myc (1:1000) (#13987s, Cell Signaling Technology).