Fig. 3

SETD1A activates the Wnt/β-catenin pathway by stabilizing β-catenin. A-B, MYC, CCND1 and nuclear β-catenin levels in NSCLC cells as indicated were analyzed by western blotting. C, CTNNB1 transcript levels in NSCLC cells as indicated was analyzed by qRT-PCR. ns, not significant. D, Subcellular localization of SETD1A and β-catenin in NSCLC cells was analyzed by confocal laser scanning microscope. The subcellular distribution of SETD1A and β-catenin was quantified by Image J software. E, The interaction between SETD1A and β-catenin in PC9 cells was analyzed by CoIP assay. F-G, β-catenin stability in the SETD1A knockdown and negative control group PC9 cells was analyzed by CHX chase assay. H, Ubiquitination of β-catenin in PC9 cells was analyzed by CoIP assay following SETD1A knockdown. I, Two-step co-immunoprecipitation of the complex containing SETD1A, β-catenin and PKA is shown. HEK293T cells were cotransfected with the Flag-tagged SETD1A plasmid and HA-tagged β-catenin plasmid. The first immunoprecipitation was performed using anti-FLAG M2 beads. The complex was eluted using 3 × Flag peptide followed by the second step of co-immunoprecipitation with anti-HA antibody. Then the protein samples were analyzed by western blotting with anti-Flag, anti-HA and anti- anti-PKAα cat antibody. Data are shown as means ± SD.