Figure 3

Moderate overexpression of phospho-relay components does not activate the HOG pathway. A Homozygous GEV diploid strains with a single inducible copy of a phospho-relay gene were grown to saturation in different concentrations of β-estradiol. The optical density (OD ₆₀₀) after 13 hours of growth is plotted. Overexpression of Pbs2 caused a growth defect at higher concentrations of β-estradiol, while no significant growth defects were observed following overexpression of relay components (beyond the defect in the wild-type due to overexpression of Gal4 target genes). Each point represents the mean and standard deviation over four replicates. B We assayed for Hog1 phosphorylation using an antibody specific to doubly phosphorylated Hog1 to confirm that moderate overexpression of phospho-relay components does not lead to activation of the HOG pathway. Overexpression of phospho-relay components using a saturating dose (10 μM) of β-estradiol did not lead to Hog1 phosphorylation. Overexpression of the positive controls Ssk22, Ssk2, and Pbs2, however, led to clear upregulation of Hog1 phosphorylation after 30 minutes of induction. Total Hog1 is shown as a loading control.