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. 2017 Sep 11;8:499. doi: 10.1038/s41467-017-00547-y

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© The Author(s) 2017

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 — Cas5 becomes activated by dephosphorylation in response to cell wall stress. a Cas5 localizes to the nucleus in response to caspofungin. Cells were fixed and Cas5 (red) was detected by indirect immunofluorescence using α-HA antibody and α-mouse IgG-Cy3. Nuclei (green) were visualized by DAPI staining. Scale bar represents 5 μm. b Cells were subcultured in rich medium to log phase and fixed. HA-tagged Cas5 (red) and nuclei (green) were visualized as in a. Scale bar represents 5 μm. c CAS5 is induced by caspofungin. The transcript level of CAS5 was monitored by qRT-PCR and normalized to GPD1. Error bars represent standard deviation (s.d.) from the mean of triplicate samples. Significance was measured with an unpaired t test in GraphPad Prism (****P < 0.0001). d Levels of Cas5 were monitored by western blot and detected with an α-HA antibody. Actin was detected with an α-β-actin antibody as a loading control. Full blots are shown in Supplementary Fig. 2b. e Cas5 is post-translationally modified upon cell wall stress treatment. Cells were grown to log phase and subsequently treated with 125 ng/ml of caspofungin or 50 μg/ml of calcofluor white for 1 h. Total protein was resolved by SDS-PAGE and the blot was hybridized with an α-HA to monitor Cas5 migration. Full blots are shown in Supplementary Fig. 2c. f Cas5 migration and actin detection were monitored as part d. Full blots are shown in Supplementary Fig. 2d. g Protein lysates were subjected to two-dimensional gel electrophoresis coupled with western blotting. Cas5 was detected with an α-HA antibody. h Cas5 is phosphorylated in the absence of stress. Cas5 migration was monitored by western blot and detected with an α-HA antibody. Treatment of protein lysate with lambda phosphatase resulted in a faster migrating band. Full blots are shown in Supplementary Fig. 2e. i Cas5 is required for caspofungin (CF)-induction of cell wall genes. Transcript levels of ECM331 and PGA13 were monitored by qRT-PCR and normalized to GPD1. Error bars represent s.d. from the mean of triplicate samples. Significance was measured with a Tukey’s multiple comparisons test in GraphPad Prism (****P < 0.0001, **P < 0.01)