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. 2020 May 1;5:52. doi: 10.1038/s41392-020-0138-6

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — The Wnt3a-induced increase in Bcl-3 protein relies on GSK-3 kinase activity. a Immunoblot for Bcl-3 and β-catenin in HCT116 and 293 T cells treated with 100 ng/ml Wnt3a for the indicated times (left). q-RT-PCR analysis of Bcl-3 mRNA expression levels in HCT116 and 293 T cells treated with 100 ng/ml Wnt3a for the indicated time points (right). b HCT116 cells were treated with cycloheximide (CHX, 50 mg/ml) for 12 h and 100 ng/ml Wnt3a for 4 h. Then, total cell lysates were analyzed by immunoblots for Bcl-3 and β-catenin. c Immunoblot analyses of immunoprecipitated Bcl-3 for the presence of K48- and K63-linked ubiquitin after MG-132 and Wnt3a stimulation. * Represents the heavy chain band. d Immunoblot analyses of Bcl-3, p-Bcl-3 (Ser394/398), and β-catenin after Wnt3a stimulation for the indicated time points. e The normalized Bcl-3 and p-Bcl-3 (Ser394/398) protein levels detected in d. f Co-IP analyses of Flag-tagged Bcl-3 (Bcl-3-Flag) and HA-tagged GSK-3 (GSK-3-HA) expressed in 293 T cells. After treatment with Wnt3a for the indicated times, cell lysates were subjected to IP with anti-Flag antibody. g Immunofluorescence staining of Bcl-3 (red) and β-catenin (green) in HCT116 cells with or without Wnt3a stimulation for 2 h. The nuclei were stained using DAPI (blue). ImageJ software was used to quantify the results. h Cytoplasmic and nuclear levels of Bcl-3 and β-catenin in HCT116 cells treated with 100 ng/ml Wnt3a for the indicated times were analyzed by immunoblot. The results were quantified by ImageJ software. The relative protein levels of β-catenin and Bcl-3 in the cytoplasm were normalized to GAPDH levels, and the relative protein levels of these two proteins in the nucleus were normalized to Lamin A levels