Anti active β catenin

RIPA buffer mixed with protease inhibitor (Thermo Fisher Scientific, USA) was used to lyse cells for harvest of total protein. The protein was separated by 4–20% Precast-Gel (Solarbio, China) and transferred to a PVDF membrane (Millipore, USA). Then 5% bovine serum albumin was used to block the membrane for 1 h at room temperature, and incubated with primary antibody overnight at 4℃. The following primary antibodies were used: anti-Flag (Origene, USA), anti-SATB2 (Abcam, USA), anti-β-catenin, anti-active β-catenin (Cell Signaling Technology, USA), anti-β-actin (Zhongshanjinqiao, China), anti-RUNX2 (Biorbyt, England), anti-JHDM1D (Abcam, USA), anti-DKK1 (Santa Cruz, USA), anti-H3K9me2, anti-H3K27me2 (Abcam, USA). HRP-conjugated secondary antibody (1:10,000; Zhongshanjinqiao, China) was then used to incubate the membrane for 1 h at room temperature. At last, we used ECL and Super Signal detection reagents (Thermo Fisher Scientific, USA) to detect the membrane.

Cell lysates were prepared as described.20 Soluble proteins were analysed by immunoblotting with anti‐MUC1‐C (ThermoScientific), anti‐active β‐catenin (Cell Signaling Technologies), anti‐survivin (Cell Signaling Technologies) and anti‐β‐actin (Sigma). Antigen‐antibody complexes were visualized by enhanced chemoluminescence (ECL; Amersham Biosciences). Densitometry analysis was performed used image J software.

Tissues and cells were homogenized. Solubilized proteins in lysis buffer (0.1 M Tris–HCl (pH 6.8), 4% SDS, 20% glycerol, 12% 2‐mercaptoethanol, and BPB) were subjected to SDS–PAGE, and proteins were electrotransfered to nitrocellulose membranes. Immunodetection was carried out using an ECL kit (GE Healthcare) according to the manufacturer's protocol. Antibodies used were as follows: anti‐β‐catenin (1:5,000, BD, #610154), anti‐P‐β‐catenin (1:2,000, Cell Signaling Technology, #4176), anti‐active‐β‐catenin (1:2,000, Cell Signaling Technology, #19807), anti‐PTEN (1:2,000, Cell Signaling Technology, #9559), anti‐P‐PTEN (1:2,000, Cell Signaling Technology, #9554), anti‐AKT (1:1,000, Cell Signaling Technology, #9272), anti‐P‐AKT (1:1,000, Cell Signaling Technology, #9271), anti‐IGF‐1Rβ (1:1,000, Cell Signaling Technology, #3018), anti‐P‐IGF‐1Rβ (1:1,000, Cell Signaling Technology, #4568), and anti‐HSC70 (1:2,000, Santa Cruz Biotechnology, #sc7298).

Tissue sections or cells were fixed, washed and blocked, and then incubated with the following primary antibodies: anti-Nestin (1/200; Abcam), anti-NF200 (1/200; Abcam), anti-MBP101 (1/200; Abcam), anti-Active β-catenin (1/200; Cell Signaling Technology), anti-DSPP (1/200; Zen Bioscience), anti-DMP1 (1/200; Santa Cruz Biotechnology), anti-CD31 (1/200; Zen Bioscience), and anti-VEGF (1/200; Abcam). The goat anti-rabbit 488 (1/200; Invitrogen) and goat anti mouse 488 (1/200; Invitrogen) secondary antibodies were used. Sections or cells were counterstained with DAPI, mounted with fluorescent mounting media and visualized using a fluorescence confocal microscope (Olympus FV1200, Olympus). Accordingly, quantitative analysis of the staining was conducted by measuring the average optical density using the ImageJ software. At least 3 replicates were performed.

Cells were grown to 30-40% confluence on coverslips. Cells were briefly washed with 1x PBS twice and fixed with 4% formaldehyde for 15 minutes at room temperature. Subsequently, cells were permeabilized with 100% methanol at −20°C for 10 minutes and blocked for 1 hour with 5% goat serum (Catalog no. 9023, Sigma Aldrich) in 1x PBS-Triton (0.3%). Cells were then incubated with 1:200 anti-β-catenin antibody (Catalog no. 2677, Cell Signaling) or 1:200 Anti-Active-β-catenin (ABC) diluted in blocking buffer overnight at 4°C. This was followed by incubation with AlexaFluor® 555 goat anti-mouse antibody (Catalog no. A21422, Invitrogen) for visualization. At the completion of secondary antibody incubation, cell nuclei were stained with 300 nM 4′, 6-diamidino-2-phenylindole (DAPI) for 7 minutes (Catalog no. D1360, Invitrogen). Coverslips were briefly rinsed with PBS and mounted on glass slide using Prolong antifade (Catalog no. P7481, Invitrogen). Washes were carried out 3 times, 5 minutes each with PBS after fixation, permeabilization and primary antibody incubation. Imaging was carried out at 40X magnification (oil immersion) using Carl Zeiss Laser Scanning Microscope and image processing was carried out using LSM image browser software.

Fresh intestinal tissues were washed in 1 mL of ice-cold PBS several times. Tissue and frozen steel beads were placed into a 2 mL centrifuge tube, and tissues were homogenized and lysed in lysis buffer with a protease and phosphatase inhibitor cocktail (Sigma). After clearing by centrifugation, protein concentrations in the supernatant were determined using a spectrophotometer (Thermo Fisher). Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore). Significant results are shown, and similar results were obtained in at least three independent experiments. The primary antibodies were anti-β-catenin (BD Biosciences, lot 610154), anti-active β-catenin (Cell Signaling, lot D13A1), anti-psmad1/5 (Cell Signaling, lot 41D10); and anti-math1 (Santa Cruz, lot sc-136173). Proteins were detected by ECL WB detection regent (Thermo Fisher).