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. 2024 Feb 15;81(1):91. doi: 10.1007/s00018-024-05148-9

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© The Author(s) 2024

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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Fig. 2 — CircYthdc2 encodes a 170 amino acid (aa) novel protein, Ythdc2-170aa. A Upper panel, the putative ORF in circYthdc2. Lower panel, the sequences of the putative ORF are shown. B The putative IRES activity in circYthdc2 was tested. C Left panel: Full-length or truncated circYthdc2 IRES sequences were cloned before GFP as indicated to construct reporter plasmids. Right panel: The empty vector, and full-length or truncated IRES vector were cotransfected with si-eif4E into HEK293 cells, and GFP signals were detected. D Schematic diagram of FLAG-circYthdc2, Linear-FL-Ythdc2-AG, and Linear-FLAG-Ythdc2-170aa plasmid construction. E Upper panel: The putative Ythdc2-170aa amino acid sequences and antibody generation region were shown as indicated to produce the Ythdc2-170aa antibody. The red amino acids were distinctly formed by the circYthdc2 junction. Lower: FLAG tag antibody was used to detect Ythdc2-170aa expression in MKC cells transfected with the vectors mentioned in Fig. 2D. In addition, Ythdc2-170aa antibody was used to detect Ythdc2-170aa expression in MKC cells after SCRV infection F FLAG-circYthdc2, Linear-FL-Ythdc2-AG, and Linear-FLAG-Ythdc2-170aa plasmids were transfected into MKC cells. Immunofluorescence staining using anti-Flag was performed to show the Ythdc2-170aa cellular localization. Original magnification is 630; all data represent the means ± SE from three independent triplicate experiments. *, p < 0.05; **, p < 0.01