Fig. 4.

ISG12a induces the proteasomal degradation of β-catenin. a qRT-PCR analysis of expression of ISG12a, CTNNB1, Axin, CKIα, APC, and GSK3β mRNAs in Huh7 cells with ISG12a knockdown. b RNA sequencing showing the expression change of mRNAs associated with the canonical Wnt/β-catenin signaling pathway in Huh7 cells with ISG12a knockdown. The scale bar shows the expression change of genes by the log₂(value+1) method. c–e Immunoblots showing β-catenin levels in Huh7 cells with ISG12a knockdown after treatment with 100 μg/ml CHX for different times (c) or 25 mM NH₄Cl (d) or 25 μM MG132 (e) for 6 h. CHX cycloheximide. f Co-IP of ubiquitinated β-catenin (HA) in HEK293T cells overexpressing ISG12a. Cell lysates were immunoprecipitated with an anti-Flag antibody and examined for HA levels by immunoblotting. g Immunoblots showing levels of Flag, β-catenin, GSK3β, Axin, and Cyclin D1 in Huh7 cells after treatment with 25 μM MG132 for 6 h. ISG12a-silenced cancer cells were further transfected with p3×Flag-ISG12a or an empty vector for 48 h. Experiments were independently repeated two (c, d) or three (a, e–g) times, with similar results. Two-sided Student’s t tests were performed to analyze the results, and data are presented as means ± SD of three biological replicates. ***p < 0.001