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. 2022 Jul 8;13(7):590. doi: 10.1038/s41419-022-05023-0

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© The Author(s) 2022

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 — A, B Overexpression or silencing of NONO decreased or increased the ubiquitination level of SAMHD1, respectively. A HA-tagged SAMHD1 was cotransfected with Myc-tagged NONO and Flag-ubiquitin or empty vector into 293 T cells for 24 h. The SAMHD1 protein was enriched by IP, and the ubiquitination level of SAMHD1 was determined by western blotting. B 293 T cells transfected with NONO or negative control siRNAs for 24 h were then cotransfected with HA-tagged SAMHD1 and Flag-ubiquitin or empty vector for another 24 h, and the ubiquitination level of SAMHD1 was examined by IP followed by western blotting. C The NONO truncation mutant with deletion of the RRM domain did not affect the ubiquitination of SAMHD1. HA-tagged SAMHD1 was cotransfected with wild-type or mutant Myc-NONO and Flag-ubiquitin into 293 T cells for 24 h, and the ubiquitination level of SAMHD1 was examined by IP followed by western blotting. D Deletion of amino acids 115-562 in SAMHD1 abrogated the influence of NONO on SAMHD1 ubiquitination. Wild-type or mutant Myc-tagged SAMHD1 was cotransfected with HA-NONO and Flag-ubiquitin or empty vector into 293 T cells for 24 h, and the ubiquitination of SAMHD1 was evaluated using IP followed by western blotting.