Anti active β catenin antibody

IHC was prepared and performed as previously described.^{23 (link),26 (link)} Samples were respectively incubated with anti-(P)RR antibody^{27 (link),28 (link)} (1: 3000), anti-active β-catenin antibody (1: 1000; Cell Signaling Technology, Danvers, MA, USA, catalog #8814), or anti-c-Myc antibody (1: 50; Abcam, Eugene, OR, USA, catalog #ab39688), as primary antibody for 1 h. Staining was considered positive when brown granules were observed in cells. Rabbit serum from which the (P)RR antibody was isolated served as the negative control to stain colorectal tissues using the same protocol as the (P)RR antibody. As previously described,^{29 (link),30 (link)} the intensity of positive staining in cancer lesions was graded from 1 to 4 (1 as the weakest intensity and 4 as the strongest intensity) by four individuals, independently. The protein expression level in each cancer lesion was determined based on the average intensity score: 1 ≤ weak < 2, 2 ≤ middle ≤ 3, and 3 < strong ≤ 4.

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.

Western blotting analysis was performed as previously described.^{23 (link)} Loading amount of the whole cell extract protein for each sample was 50 μg. The primary antibodies used were anti-(P)RR antibody (1:1000),^{27 (link),28 (link)} anti-Wnt3 antibody (1: 500; Abcam, catalog #ab32249), anti-total LRP6 antibody (1:800; Cell Signaling Technology, catalog #3395), anti-phosphorylated LRP6 antibody (Ser 1490, 1:800; Cell Signaling Technology, catalog #2568), anti-active β-catenin antibody (1:1000; Cell Signaling Technology, catalog #8814), anti-Cyclin D1 antibody (1:1000; Cell Signaling Technology, catalog #2922), anti-c-Myc antibody (1:1000; Cell Signaling Technology, catalog #5605) and anti-β-actin antibody (1:1000; Sigma-Aldrich, catalog #A5441). β-Actin served as internal control. Consistent results were obtained from at least three times of independent experiment.