FIG. 2.

Western blot analysis of phospho-Hog1 and total Hog1 from S. cereviseae strains using (A) anti-p38 antibody, raised to a synthetic doubly phosphorylated segment of the mammalian protein P38 corresponding to T180/Y182 (Cell Signaling Technology), or (B) anti C-terminal Hog1 antibody (Santa Cruz Biotechnology). S. cerevisiae ATCC 201388/pYES2 (SC/pYES2) and S. cerevisiae ATCC 201388/pYES2-HIK1 (SC/pYES2-HIK1) are described in Table 1. SC Δ hog1 is an isogenic derivative of ATCC 201388 but carries a deletion of the HOG1 gene (14). S. cerevisiae strain ATCC 201388 was transformed with either pYES or pYES-HIK1, as described in Table 1, or disrupted for HOG1 (Δhog1), as described in reference 14. Cells from an overnight culture in SD/−URA were resuspended in fresh SD/−URA and grown for 4 h. The cells were treated for 10 min with NaCl (0.5 M) or ambruticin (25 ppm) or were not treated (NT) and cell extracts were prepared. Soluble protein was used for Western blot analysis. Antibody binding was visualized using a horseradish peroxidase-conjugated secondary antibody employing detection with ECL Plus (Amersham).