Comprehensive Protein Analysis Workflow

We performed IHC, western blotting and IP according to previously described methods [8 (link)]. Especially, the key preconditions of IP experiment are: 1. the basal loading amounts of total protein, which is directly extracted from cells, are equal in each group; 2. the antibodies used for IP and immunoblotting (IB) are of equal amounts in each group. Primary antibodies used were: anti-(P)RR antibody (Sigma-Aldrich, Allentown, PA, USA, catalog #HPA003156), anti-active β-catenin antibody (Cell Signaling, Technology, Danvers, MA, USA, catalog #8814), anti-c-Myc antibody (Abcam, Eugene, OR, USA, catalog #ab39688), anti-Wnt3 antibody (Abcam, Eugene, OR, USA, catalog #ab32249), anti-NEDD4L antibody (Cell Signaling Technology, Danvers, MA, USA, catalog #4013), anti-flag antibody (HuaAn Biotechnology, Hangzhou, China, catalog #0912-1) and anti-GAPDH antibody (HuaAn Biotechnology, Hangzhou, China, catalog #ET1601-4). GAPDH served as internal control. IF was performed as previously described [17 (link)]. Primary antibodies used were: anti-Wnt3 antibody (Thermo Fisher Scientific, Waltham, MA, USA, catalog #PA5-18,516) and the same anti-NEDD4L antibody that was used in western blotting. All these antibodies were used according to the instructions provided by manufactures.