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. 2015 Jul 15;26(14):2609–2619. doi: 10.1091/mbc.E14-07-1213

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© 2015 Al-Zain et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — Analysis of GSK-3 consensus sites in Cdc6. (A) Cdc6 contains two GSK-3 consensus sites, which overlap with two CDK consensus sites. (B) GAL-CDC6-HA strains with various mutations (T368A, P369A, S372A, P373A, T39A, S43A, T39A-T368A, and T368A-S372A) were expressed with galactose-containing medium for 2 h and then blocked with nocodazole for 2 h. Cdc6 expression was then suppressed by adding glucose. Protein extracts were collected every 5 min and subjected to Western blot analysis to observe Cdc6-HA. Pgk1 was used as a loading control. GAL-CDC6-HA in mck1-deletion cells was examined using the same method. Western blotting images for WT, Δmck1, CDC6-T39A, CDC6-T368A, and CDC6-T39A,T368A were quantified. Percentage of Cdc6 protein remaining relative to time zero is shown. Results are the average of three independent experiment, and error bars indicate SD. *p < 0.05. (C) Strains with the indicated genotypes were serially diluted 10-fold, plated on yeast extract/peptone/dextrose or yeast extract/peptone/galactose plates, and incubated at 30°C for 2 d. (D) GAL-CDC6, GAL-CDC6-T368A, or GAL-CDC6-T39A-T368A was grown in raffinose-containing medium first. Cdc6p was expressed with galactose for 3 h.