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. 2021 Jul 20;12:4427. doi: 10.1038/s41467-021-24724-2

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© The Author(s) 2021

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. 8 — a HEK293T cells were transfected with Flag-tagged-M2 from different IAV strains. Expression of M2 was examined in western blots. b Effect of MARCH8 on the expression of M2 proteins from different IAV strains. The HEK293T cells were transfected with different M2 molecules and Vector, MARCH8 or W114A. M2 and MARCH8 were detected with specific antibodies. c Ubiquitination of M2 from different IAV strains by MARCH8. The HEK293T cells were transfected with M2, MARCH8 and HA-Ub. Cell lysates were subject to IP with anti-flag antibody, and the IP and input were analyzed by western blotting with antibodies against the indicated targets. d HEK293T cells were transfected with control, MARCH8 or W114A, then infected with WSN virus, PR8 virus, pdm09 virus or H3N2 virus for 24 h (MOI = 0.1). Viral titers in cell culture supernatants were determined by plaque assay. Data shown are the means ± SD (n = 3 biological replicates). **P < 0.001; n.s., nonsignificant; unpaired two-tailed Student’s t-test, without any adjustments for multiple comparisons. e M2 protein amino-acid sequence logo of IAV isolates. The K78 residues are marked as red. M2 sequences were downloaded from GenBank using the “collapse identical sequences” option. Logos were generated using the WebLogo3 online tool.