Western blot analysis of phospho beta catenin was performed by loading 30 µg of HT29 WT untreated (Lane 1), HT29 WT EGF treated (Lane 2), HT29 Cas9 untreated (Lane 3), HT29 Cas9 EGF treated (Lane 4), HT29 beta catenin knockout untreated (Lane 5) and HT29 beta catenin knockout EGF treated (Lane 6) whole cell lysate. The blot was probed with Phospho-beta Catenin (Ser552) Polyclonal Antibody (Product # MA5-17154, 1:1,000 dilution) and Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4,000 dilution. Loss of signal upon CRISPR mediated knockout (KO) confirms that antibody is specific to phospho beta catenin. A non-specific band was also observed at ~52 kDa with the above testing conditions.