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. 2024 Feb 15;84(9):1443–1459. doi: 10.1158/0008-5472.CAN-23-2268

Figure 2.

Figure 2. Analysis of β-catenin signaling in cell clones with endogenous knockout of the GSK3β and β-catenin domains. HEK293T and SNU449 AXIN1-repaired cells (12) were used to knockout the GSK3β-binding domain encoded by exon 5. We also identified one HEK293T clone with an endogenous knockout of the β-catenin–binding region. A, Immunoblot using an N-terminal AXIN1 antibody (cat. #3323, Cell Signaling Technology) to show expression of the shortened AXIN1 proteins. β-Actin served as a loading control. B, qRT-PCR assay to demonstrate the mRNA expression level of AXIN2 (in triplicate, n = 2 independent experiments). Expression levels were depicted relative to the housekeeping gene GAPDH. The value for the WT control was arbitrarily set to 1. C, A β-catenin luciferase reporter assay was performed to determine the β-catenin signaling activity in all knockout clones. Values are depicted as WRE/MRE ratios (in triplicate, n = 3 independent experiments). The value for the WT control was arbitrarily set to 1. D, siRNA-mediated knockdown of AXIN2 was performed in all knockout clones, followed by a β-catenin reporter assay. Values are depicted relative to the WRE/CMV-Renilla ratios obtained for the siControl-WT (five replicates, two independent experiments), which was arbitrarily set to 1. Data are shown as mean ± SD. Statistical significance for all experiments was analyzed using a Mann–Whitney test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Analysis of β-catenin signaling in cell clones with endogenous knockout of the GSK3β and β-catenin domains. HEK293T and SNU449 AXIN1-repaired cells (12) were used to knockout the GSK3β-binding domain encoded by exon 5. We also identified one HEK293T clone with an endogenous knockout of the β-catenin–binding region. A, Immunoblot using an N-terminal AXIN1 antibody (cat. #3323, Cell Signaling Technology) to show expression of the shortened AXIN1 proteins. β-Actin served as a loading control. B, qRT-PCR assay to demonstrate the mRNA expression level of AXIN2 (in triplicate, n = 2 independent experiments). Expression levels were depicted relative to the housekeeping gene GAPDH. The value for the WT control was arbitrarily set to 1. C, A β-catenin luciferase reporter assay was performed to determine the β-catenin signaling activity in all knockout clones. Values are depicted as WRE/MRE ratios (in triplicate, n = 3 independent experiments). The value for the WT control was arbitrarily set to 1. D, siRNA-mediated knockdown of AXIN2 was performed in all knockout clones, followed by a β-catenin reporter assay. Values are depicted relative to the WRE/CMV-Renilla ratios obtained for the siControl-WT (five replicates, two independent experiments), which was arbitrarily set to 1. Data are shown as mean ± SD. Statistical significance for all experiments was analyzed using a Mann–Whitney test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.