Expression of Hcm1‐3N‐3HA and Hcm1‐8C‐3HA over the cell cycle

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Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression Expression of Hcm1‐3N‐3HA and Hcm1‐8C‐3HA over the cell cycle. Cells were arrested in G1, released into the cell cycle, and samples taken for Western blot and flow cytometry (Supplementary Fig S8A) at 15‐min time points. Cells expressing Hcm1‐GFP or Hcm1‐3N‐GFP from the TEF1 promoter were arrested in G1 with alpha‐factor, S‐phase with HU, or mitosis with nocodazole and half‐lives compared by cycloheximide‐chase assay. Levels of Hcm1, Clb2, and cyclin‐dependent kinase 1 (Cdk1) are shown. Cell cycle arrest was confirmed by flow cytometry (Supplementary Fig S8B). Cells expressing the indicated Hcm1 mutants from the TEF1 promoter were arrested in mitosis (Supplementary Fig S8C) and half‐lives compared by cycloheximide‐chase assay. Two exposures of Hcm1 blots are shown to highlight differences in stability between the double phosphomutants. Cycloheximide‐chase assay of Hcm1(1‐107)‐GFP and Hcm1(1‐107)3N‐GFP fusion proteins in asynchronous cells. CDC53, cdc53‐1, sic1Δ CDC53, and sic1Δ cdc53‐1 cells expressing Hcm1(1‐107)‐GFP were arrested in mitosis (Supplementary Fig S8D) at the permissive temperature, shifted to 37°C for 15 min, and half‐lives compared by cycloheximide‐chase assay. Fivefold dilutions of cells with the indicated genotypes were spotted onto rich medium plates (YPD), or plates containing the indicated concentrations of benomyl. Source data are available online for this figure. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO J, Volume: 33, Issue: 9, Pages: 1044-1060, First published: 08 April 2014, DOI: (10.1002/embj.201386877)