FIG 4.

(A) Acidic pH does not induce Hog1 phosphorylation. A Phospho-Hog1 immunoblot of cells grown for 3 h to the logarithmic phase and inoculated into fresh YPD medium at pH 4, pH 7, or pH 7 with 0.5 M NaCl for 5 min is shown. A pbs2 mutant strain was induced in 0.5 M NaCl as a negative control. A parallel blot was probed with anti-PSTAIRE as a loading control. (B) Acidic pH sustains Hog1 basal phosphorylation. A Phospho-Hog1 immunoblot of overnight cells (0 h) inoculated into fresh YPD medium at pH 4 and pH 7 for 3 h is shown. Aliquots were collected every hour. At 3 h, an aliquot of cells were shifted to medium with 1 M NaCl (3N) to induce Hog1 phosphorylation. A parallel blot was probed with anti-PSTAIRE as a loading control. (C) Acidic pH inhibits PTP3 transcription. qRT-PCR of WT and pbs2 cells was performed to measure the levels of PTP3 transcript after cells were grown at pH 4 and pH 6 for 15 min. qPCR values were normalized to ACT1 for each samples, and overnight (ON) samples were set to a value of 1. (D) Hog1 phosphorylation inhibits hyphal initiation. Morphology and HWP1p-GFP expression of WT, ptp2 ptp3, and ptp2 ptp3 hog1 strains expressing a copy of HWP1p-GFP after inoculation for 2.5 h in YPD medium at pH 7 or pH 4 or supplemented with 0.5 M NaCl are shown.