Ab137827

Cells were lysed using RIPA buffer. The lysate was separated by 10−15% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). The membrane was blocked with 5% nonfat milk and incubated with anti-LEF-1 (1:1000, ab137827, Abcam), anti-β-catenin (ab16051, 1:1000, Abcam), anti-CCND2(ab230883, 1:1000, Abcam), anti-CyclinD1 (ab226977, 1:1000, Abcam), anti-MYC (ab32072, 1:1500, Abcam), anti-SOX4 (ab86809, 1:1000, Abcam), anti-Ubiquitin (ab134953, 1:1000, Abcam) and anti-GAPDH (ab8245, 1:1000, Abcam) antibodies overnight at 4 °C. Then the membrane was washed with TBST three times and incubated with the secondary antibody (Abcam, Cambridge, MA) for 1.5 h at room temperature. The ECL chromogenic solution was used to display the chemiluminescence of the bands. The Quantity One 4.4.0 software was used for densitometry determination.

Mammalian cells were lysed 42 h after transduction with lysis buffer (0.5% Triton X100, 50 mM Tris–HCl, 100 mM NaCl, pH 7.5). The supernatant was used for western blotting after sonification and centrifugation. Rabbit anti-GFP (Abcam ab137827; diluted 1:2500 in 3% BSA/TBST) and mouse anti-tubulin (Sigma T5168; diluted 1:2,500 in 3% BSA/TBST) were used as primary antibodies. As secondary antibodies, alkaline phosphatase-coupled goat anti-rabbit and anti-mouse as well as donkey anti-human IgGs (Southern Biotech, diluted 1:10,000 in TBST) were used, followed by chemiluminescence detection. Quantification was performed with ImageJ. Original western blots are shown in Supplementary Fig. 10.