Anti phospho β catenin antibody

HEC-1B and Ishikawa protein was extracted using the RIPA Lysis Buffer System (Santa Cruz Biotechnology, Dallas, TX, USA). Five micrograms of protein (whole cell extracts) was subjected to sodium dodecyl sulfate-poly acrylamide gel electrophoresis (10% acrylamide gel). Following SDS-PAGE, proteins were transferred onto Hybond-P Polyvinylidene Difluoride membrane (GE Healthcare, Little Chalfont, UK). Anti-phospho-β-catenin antibody (#4176) was purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin was purchased from Sigma, and anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-Horseradish peroxidase was purchased from Wako. Antibody–protein complexes were detected on the blots using ECL Plus Western blotting detection reagent (GE Healthcare), and the protein bands were visualized with a LAS-4000 image analyzer (Fuji Photo Film, Tokyo, Japan). The relative intensity values of the protein bands were measured by the use of an imaging analyzer, Lumina Vision (Mitani Corp., Fukui, Japan). The expression level for phospho-β-catenin was summarized as a ratio of GAPDH.