Axin1

Whole cell lysates were obtained from DPSCs cultured under control conditions or stimulated with ferutinin (10 μg/mL in αMEM) or CHIR-98014 (50 nM) for 12, 24, and 48 h. Protein was quantified by colorimetric assay using the Bradford method (Bio-Rad, Hercules, CA) and the proteins were separated in a polyacrylamide gel. Briefly, a polyacrylamide gel was cast and denatured proteins (20 µg) were loaded and separated through the gel by electrophoresis; a protein ladder was loaded as a marker (Sigma, St. Louis, MO). The proteins were transferred from the gel to a 0.45 µm nitrocellulose membrane (Bio-Rad) at 4 °C. The membrane was blocked for 1 h at room temperature (RT) with a blocking buffer composed of 5% nonfat milk in TBS-Tween-20 (TBST) (Boston BioProducts, Ashland, MA). The membrane was washed and incubated with primary antibody against LRP6, Dvl3, Axin1, Naked1, β-catenin, GAPDH (Cell Signaling, Danvers, MA), and GSK3 (Santa Cruz Biotechnology, Dallas, TX) (1:1000 diluted in a solution of 5% BSA in TBST) for 2 h. The membrane was washed, then incubated in secondary antibody (1:3000 in a solution of 5% milk in TBST) (Cell Signaling). The membrane was then washed, placed in the cassette holder and incubated briefly in chemiluminescent substrate (Sigma). Films were then exposed and developed. Densitometric quantification of bands was performed using ImageJ software (NIH).

Intestinal tissues from mice were isolated and sonicated in lysis buffer (1% Triton X-100, 150 mmol/L NaCl, 10 mmol/L Tris pH 7.4, 1 mmol/L EDTA, 1 mmol/L EGTA pH 8.0, 0.2 mmol/L sodium ortho-vanadate, and protease inhibitor cocktail), as previously described [12 (link)]. MEFs, HCT116, and HT29 cells rinsed twice with ice-cold PBS, were lysed and sonicated in protein loading buffer (50 mmol/L Tris, pH 6.8, 100 mmol/L dithiothreitol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol). Primary antibodies to mouse VDR, β-actin (Sigma-Aldrich, Milwaukee, WI, USA), and Axin1 (Cell Signal, Beverly, MA, USA) were used, and were visualized by ECL Relative abundance of protein was determined using Quantity One software (Bio-Rad, Hercules, CA).