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. 2018 Oct 17;9:4308. doi: 10.1038/s41467-018-06767-0

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© The Author(s) 2018

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. 5 — Dma1 and Dma2 promote Nud1 ubiquitination and inhibit recruitment of MEN factors to SPBs. a–c Ni-NTA pulldown assays were carried out using cell extracts from strains with the indicated genotypes expressing Nud1-3PK at endogenous levels and overexpressing either untagged ubiquitin or His-tagged ubiquitin from the CUP1 promoter. Nud1 ubiquitination was revealed by western blot using an anti-PK antibody. DMA2 was overexpressed for 30 min by addition of 1% galactose to raffinose-containing medium (b). For the time-course experiment in c cells were grown in -Trp medium (mock and cyc), arrested in G1 by alpha factor in YEPD and then released in fresh YEPD (time 0). At the indicated times cells were collected for Nud1 ubiquitination assays and tubulin immunofluorescence. Mock: Ni-NTA pulldown from cells extracts of cells expressing Nud1-3PK and untagged ubiquitin. Cyc: cycling cells. d Cells expressing Spc42-mCherry along with a specific MEN factor tagged with GFP/eGFP were arrested in G1 by alpha factor and then released in fresh medium at 25 °C to enrich cells in anaphase. Cells were fixed at different time points for quantifying the relative fluorescence of MEN factor vs. Spc42-mCherry in anaphase (see Methods). n ≥ 60. Statistical significance of differences was assessed by two-tailed t test, assuming unequal variances (^*p < 0.05; ^**p < 0.01; ^***p < 0.001; n.s.: not significant). e Wild-type and GAL1-DMA2 cells expressing Mob1-GFP were imaged at 30 °C every 4 min in SD-raffinose/galactose. Fluorescent dots represent SPBs, while the arrowhead indicates in the transient appearance of Mob1 at the bud neck of wild-type cells. Scale bar: 5 µm. f Wild-type and GAL1-DMA2 cells expressing Nud1-3PK were grown in YEPR, arrested in G1 with alpha factor and released in fresh YEPRG medium after 30 min induction with galactose. Cells were collected at the indicated times after release (time 0) for FACS analysis of DNA contents (Fig. S11b), in situ immunofluorescence of tubulin and for western blot detection of Nud1-pS78 and Nud1-3PK. Cyc: cycling cells