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. 2013 Feb 1;24(3):409–420. doi: 10.1091/mbc.E12-07-0516

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© 2013 Yerko 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 4: — Mutational analysis of the essential role of the PH^Ste5 domain in pheromone response. (A) β-Galactosidase assay (top) and Halo assay (bottom) of yeast cells (ste5Δ) transformed with STE5 alleles carrying mutations in the PH domain for their ability to induce pheromone-dependent transcriptional activation of mating-specific reporter gene and cell cycle arrest. (B) Pull-down assay with bacterially expressed GST-RBL^Ste11- and His-tagged PH^Ste5-domain mutants. Western blot analysis of pull-down and input of PH^Ste5-domain mutants was carried out with anti-His antibody. (C) Activated STE11 allele bypasses the mating defect of the PH^Ste5-domain mutant. Yeast cells (ste5Δ) cotransformed with either STE5 or STE11 allele or vectors in combination as indicated were assayed for their ability to mate, and the mating products were revealed on selective medium (right).