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. 2023 Aug 3;13:12584. doi: 10.1038/s41598-023-38272-w

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© The Author(s) 2023

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Western blot analysis for POU4F3 wild-type, frameshift, missense variants by transient transfection at HEK293T cell. (a) Expressions of POU4F3 wild-type and mutants were detected by western blotting in HEK 293T cells. Molecular weight of wild-type and mutant proteins (p.Leu248Pro, p.Phe293Leu, and p.Val318Met) are 36 kDa, whereas molecular weight of truncated mutant protein (p.Ala189SerfsTer26) is 21 kDa. The immunoblots are representative of independent repetitive experiments. LacZ is used as a transfection control. (b) The bands intensity was quantified by Image J. The band intensity was normalized to β-actin. Intensity data was presented as means ± standard deviations from two independent plots in a triplicate manner. Consequently, the sample size for each experiment was six. (c) Comparison of the stability of wild-type and mutant POU4F3 using protein stability assays in the transient overexpression system. HEK293 cells, overexpressing POU4F3, were treated with cycloheximide (80 µg/ml) for up to 3 h to block the general translation. The CHX chase assay experiment was performed once, with three measurements taken during the process. As a result, the sample size for each experiment was three. WT, wild type; LacZ, transfection control; β-actin, loading control; ns, no statistical significance; *p < 0.05, ***p < 0.001, one-way ANOVA with Bonferroni comparisons. The original immunoblots (uncropped, full length membranes with membrane edges visible, and standard protein size markers and expected molecular weight labeled) matched to the cropped versions (this figure) were all provided in Fig. S3.