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. 2020 Sep 24;11:4845. doi: 10.1038/s41467-020-18542-1

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© The Author(s) 2020

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 A time-resolved interactome of M97 during MCMV infection (Supplementary Fig. 6). Enrichment ratios for proteins co-purifying with HA-M97 at 12 or 36 h post infection in a scatterplot. The cut-off at SILAC fold-change 1.5 is indicated by a red dotted line. The mean of n = 3 replicates is depicted. b–f Serum-starved 3T3 fibroblasts were infected with the indicated MCMV-HA-M97 variants. b The expression of Cyclin A and of selected immediate-early, early and late MCMV gene products was monitored by immunoblot analysis. Affinity purification of the indicated HA-M97 variants by anti-HA-coupled paramagnetic microbeads (c) or reverse co-IP (d) confirms RXL/Cy-dependent interaction with Cyclin A–CDK2. Assays for M97 (e) and Cyclin A (f) enzymatic activity. Lysates from MCMV-infected 3T3 fibroblasts were subjected to anti-HA-IP (e) or Cyclin A-IP (f) and used as input material for γ-P32-ATP kinase assay. Recombinant Histone H1 was used as RXL/Cy-independent Cyclin A substrate and autophosphorylation of M97 was measured for the M97 kinase assay. Incorporation of γ-P32-ATP was visualized by autoradiography. The immunoblots (b–f) and autoradiograph (f) are representative of three independent experiments with similar results.