Rabbit anti axin1

Total cell lysates were obtained using radioimmunoprecipitation assay buffer (Solarbio, Beijing, People’s Republic of China) containing 1:100 phenylmethylsulfonyl fluoride and phosphatase inhibitors. Total cellular proteins were extracted and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad Laboratories Inc., Hercules, CA, USA) and transferred to 0.22 μm nitrocellulose (NC) membranes (Sigma-Aldrich Co., St Louis, MO, USA). The membranes were blocked with 5% nonfat milk at room temperature for 2 hours and then incubated overnight at 4°C with rabbit anti-FZD8 (Sigma-Aldrich Co.), rabbit anti-LRP6 (Santa Cruz Biotechnology Inc., Dallas, TX, USA), rabbit anti-Axin1 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-c-myc (Cell Signaling Technology), rabbit anti-cyclin D1 (Cell Signaling Technology), rabbit anti-β-catenin (Sigma-Aldrich Co.), rabbit anti-E-cadherin (Cell Signaling Technology), or mouse anti-β-actin antibodies (California Bioscience, Coachella, CA, USA). After incubating with IRDyeTM800 (green)- or IRDyeTM700 (red)-conjugated affinity-purified antirabbit or antimouse IgG (LI-COR, Lincoln, NE, USA), specific bands were visualized. The intensity of protein bands was evaluated using a LI-COR Odyssey infrared double-fluorescence imaging system (LI-COR).