Mouse anti total β catenin

ES cells were washed with PBS, trypsinized, and collected by centrifugation. Whole cell lysates were prepared using RIPA cell lysis buffer (Sigma) containing 1:100 phosphatase inhibitor cocktail 2, phosphatase inhibitor cocktail 3 and protease inhibitor (Sigma). Samples were rotated for 30 min at 4 °C and spun at max speed for 10 min at 4 °C. The supernatant from samples was collected and protein concentration was determined by Bradford assay. Equal amounts of protein per sample were combined with Laemmli buffer, denatured for 5 min at 95 °C and subjected to SDS/PAGE separation, followed by immunoblotting. The following primary antibodies were used: rabbit anti-active β-Catenin (Cell Signaling Technology, 8814), mouse anti-total β-Catenin (BD Biosciences, 610154), rabbit monoclonal anti-Tcf1 (Cell Signaling Technology, 2203), rabbit monoclonal anti-Lef1 (Cell Signaling Technology, 2230). Mouse anti-β-ACTIN (Santa Cruz Biotechnology; sc-47778) was used as a load control. The antibodies used in this study can be found in Supplementary Table 1. Protein quantification was performed with ImageJ software from n = 3 independent biological replicates (Fig. 1e). Uncropped and unprocessed scans are provided in the Source data file (for Fig. 1e) or in Supplementary Fig. 8 for blots presented in Supplementary figures.

Western blot analyses on lysates from HCEC, HCT116, SW480, DLD1 and TOV-112D cells were performed as described [45 (link)]. The following antibodies were used: mouse anti-active β-catenin (1:2000; Millipore, Temecula, CA), mouse anti-total β-catenin (1:10,000; BD Biosciences), rabbit anti-APC (clone C-20, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-APC (clone Ab-5, 1:1000; Millipore), and mouse anti-β-actin (1:10,000; Sigma). The density of Western blotting bands was quantified using AlphaImager HP system (ProteinSimple, San Jose, CA).

Cells were collected at ~90% confluence, lysed in RIPA (20mM Tris pH 7.4, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 100mM NaCl) containing protease and phosphatase inhibitors and analyzed as previously described [11 (link)]. Luciferase were collected in Luciferase assay buffer and resuspended in 2X SDS sample buffer. The following antibodies were used: mouse anti-MAMDC4 1:3 (Wilson Lab), rabbit anti-actin (cat. 4970; CST), mouse anti-total β-catenin (cat. 610153; BD Biosciences), rabbit anti total β-catenin (cat. 8480; CST) rabbit anti-non-phospho (active) β-catenin (cat. 19807; CST), rabbit anti-Axin1 (cat. 2074; CST), rabbit anti-LRP6 (cat. 2560; CST), rabbit anti-phospho-LRP6 (Ser 1490) (cat. 2568; CST), rabbit anti-total GSK (cat. 12456; CST), rabbit anti-phospho-GSK3β (Ser 9) (cat. 5558; CST), rabbit anti-DVL2 (cat. 3224; CST), rabbit anti-MST (cat. 3682; CST), rabbit anti-LATS (cat. 3477; CST), rabbit anti-phospho-LATS (cat. 8654; CST), rabbit anti-YAP (cat. 14074; CST), rabbit anti-phospho YAP (cat. 13008; CST), rabbit anti AMOT (cat. 24550–1; ProteinTech). Immunoblots were imaged using a Licor-Odyssey infrared imager. Protein expression was normalized to actin levels.