Nonphosphorylated β catenin

CHIR 99021 was obtained from Tocris Bioscience (Minneapolis, MN). The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo-lium (MTT) assay kit was purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum, Dulbecco's modified Eagle's medium (DMEM), penicillin, and streptomycin were purchased from Hyclone Laboratories, Inc. (Logan, UT). The antibodies for phosphorylated GK-3β (S9), GSK-3β, β-catenin, nonphosphorylated β-catenin, Smad2, phosphorylated Smad2 (Ser465/467), Smad3 (Ser423/425), and phosphorylated Smad3 were purchased from Cell Signaling Technology (Beverly, MA). The antibodies for phosphorylated GSK-3β (Y216) and fibronectin were purchased from BD Bioscience (San Jose, CA). Collagen Iα and β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody for α-smooth muscle actin (α-SMA) was purchased from Sigma-Aldrich.

AML cells (MV4-11 and MOLM-13) were drugged for 24 h, followed by protein extraction using RIPA buffer (Cell Signaling Technology) supplemented with PMSF. Cell pellets were incubated in RIPA buffer with constant agitation for 30 min, followed by sonication. Protein lysate was quantified using Pierce™ BCA Protein Assay Kit (ThermoFischer Scientific). 30 µg of protein was loaded into a Mini-PROTEAN TGX Stain-Free Precast Gel (Bio-Rad) and ran at 120 V for 1–2 h. Blots were developed using a ChemiDoc Chemiluminescence Imaging System (Bio-Rad) or by the Odyssey CLX Fluorescence Imaging System (Li-COR). Antibodies used in the presented studies purchased from Cell Signaling Technology included total β-catenin (#8480S), phosphorylated β-catenin (#9561S), non-phosphorylated β-catenin (cat#8814S), and β-actin (#3700S). Antibodies purchased from Abcam include LRP6 (ab134146) and phosphorlated-LRP6 (ab76417).