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. 2011 Sep 1;22(17):3253–3262. doi: 10.1091/mbc.E11-04-0314

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© 2011 Frieser 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 2: — Cdc24 is degraded upon mating type–dependent dimorphic switching. (A) Localization of Cdc24 was followed by constitutive expression of Cdc24-GFP fusion protein in strains AB31 and AB31Δrac1. These strains express the active bE1/bW2 heterodimer under control of the arabinose-inducible crg promoter. DIC and fluorescence micrographs were taken before induction (0 h) and 4 and 8 h after induction of bE1/bW2 expression. Bars, 10 μm. (B) Northern analysis of cdc24-GFP mRNA levels in strains AB31 and AB31Δrac1 before (0 h) and after induction (8 h) of the bE1/bW2 heterodimeric transcription factor. The ppi gene encoding peptidyl-prolyl cis/trans isomerase served as loading control. (C) Western blot analysis of Cdc24-GFP protein levels in strains AB31 and AB31Δrac1 before (0 h) and after induction (8 h) of the bE1/bW2 heterodimeric transcription factor. A monoclonal antibody against GFP was used for detection of Cdc24-GFP. Tubulin (Tub1) served as loading control. (D) The protein level of Cdc24 was determined at various time points after induction of bE1/bW2 overexpression in wild-type cells and in cells deleted for the endogenous copy of rac1. Cells were grown overnight in noninducing medium (0 h) and transferred to inducing medium. Proteins were isolated before induction and at 2, 4, 6, and 8 h after induction. (E) The protein level of Cdc24-3xHA expressed under control of its endogenous promoter was determined in strain AB31 and in conditional mutants of rac1 and cdc42 that express the GTPases under control of the crg promoter at the genomic loci. Cells were grown in liquid medium under noninducing (−) or inducing (+) conditions.