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. 2022 Apr 19;13:2079. doi: 10.1038/s41467-022-29690-x

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© The Author(s) 2022, corrected publication 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 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. 4 — a Schematic representation of the APL-16-5 molecular capture protocol and summary of ubiquitin-related candidates. b TRIM25-knockdown cells were infected with IAV A/WSN/33 in the presence of increasing amounts of APL-16-5 (2 μM or 10 μM). Virus production was determined via HEK293T-Gluc assay. TRIM25 levels were confirmed by western blot. For NC (DMSO vs. APL-16-5-2, 10 μM): (p = 0.0031 and p = 0.0003, respectively), TRIM25 KD (DMSO vs. APL-16-5-2, 10 μM): (p = 0.0695 and p = 0.0013, respectively). c TRIM25-knockout cells were transfected with increasing amounts of TRIM25 and infected with A/WSN/33 in the presence of APL-16-5 (2 or 10 μM). Virus production was determined via HEK293T-Gluc assay. TRIM25 levels were determined by western blot. For NC (DMSO vs. Ribavirin, APL-16-5-2, 10 μM): (p = 0.0017, p = 0.0005 and p = 0.0001, respectively), TRIM25 KO (DMSO vs. Ribavirin, APL-16-5-2, 10 μM): (p = 0.0020, p = 0.1457 and p = 0.1399, respectively). d HEK293T cells were transfected with increasing amounts of TRIM25 and infected with WSN/33 in the presence of APL-16-5 (2 or 10 μM). Virus production was determined via HEK293T-Gluc assay. For Ribavirin (TRIM25 0 ng vs. 200, 500 ng): (p = 0.3106 and p = 0.9464, respectively), APL-16-5 (TRIM25 0 ng vs. 200, 500 ng): (p = 0.0117 and p = 0.0118, respectively). e Western blot analysis of lysates from TRIM25-knockout cells transfected with PA DNA in the presence of increasing concentrations of APL-16-5 (2 or 10 μM). For NC (DMSO vs. APL-16-5-2, 10 μM): (p = 0.0061 and p = 0.0002, respectively), TRIM25 KO (DMSO vs. APL-16-5-2, 10 μM): (p = 0.0528 and p = 0.0507, respectively). f Immunoprecipitation and immunoblot analysis of TRIM25-knockout cells that were transfected with PA and CW7-myc-ubiquitin DNA in the presence of increasing concentrations of APL-16-5 (2 or 10 μM). b–e Data are normalized to those of the control group, which is arbitrarily set to 100%. The graph summarizes n = 3 independent experiments. Error bars show mean ± SEM (unpaired two-tailed t-test). **p < 0.01, ***p < 0.001, NS = not significant. Source data are provided as a Source Data file.