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. 2019 Jun 3;10:2426. doi: 10.1038/s41467-019-10321-x

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© The Author(s) 2019

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 — Chromatin-bound ORC3 and ORC5 are multi-mono-ubiquitylated in S-phase. a U2OS cells were co-transfected with the indicated Myc-Flag (MF)-tagged ORC subunits and HA-tagged ubiquitin. Two days post-transfection, cells were lysed in NEM-containing lysis buffer and tagged proteins were purified by anti-Flag immunoprecipitation. Expression and ubiquitin conjugation of the immunoprecipitates were monitored by western blotting, as indicated. b U2OS cells were co-transfected with the indicated His₆-Flag (HF)-tagged ORC subunits and HA-tagged ubiquitin. Chromatin (C) and soluble (S) fractions were isolated and ORC subunits were purified by nickel beads (Ni-NTA) under denaturing conditions. Purified proteins and input extracts were analysed by western blotting with antibodies against the indicated proteins. c U2OS cells were co-transfected with His₆-Flag-ORC5 (HF-ORC5) and the indicated HA-tagged ubiquitin single-lysine (K) mutants. In the 0 K mutant, all lysine residues were replaced by arginine residues. ORC5 ubiquitylation was detected by purification in denaturing conditions on nickel beads (Ni-NTA). Expression and ubiquitin conjugation of the isolated proteins were monitored by western blotting. d UbiCRest analysis of ubiquitylated ORC5. U2OS cells were transfected with Myc-Flag (MF)-tagged ORC5, without ectopic ubiquitin. Two days post-transfection, MF-ORC5 was immunoprecipitated (IP: Flag) and incubated with the indicated deubiquitylating enzymes (DUBs). Supernatants were recovered and analysed by silver staining. DUBs and released endogenous ubiquitin are indicated. Ubiquitylation was revealed by the presence of high molecular weight forms detected by western blotting against tagged ORC subunits. The ubiquitin linkage specificity of the used DUBs is indicated. e U2OS cells stably expressing Myc-Flag (MF)-tagged ORC3 or ORC5 were synchronised in mitosis by incubation with nocodazole and then released in normal growth medium for the indicated times. Tagged proteins were immunoprecipitated (IP: Flag) and ubiquitylation was assessed by anti-Myc antibody (upper panels). Synchronisation was evaluated by flow cytometry (lower panels)