FIG. 6.

Western blot demonstrating the dual phosphorylation of Hog1p upon exposure to citric acid. Soluble protein extracts were prepared from S. cerevisiae BY4741 MATa (BY4741/a) and Δhog1, Δssk1, and Δpbs2 mutants with or without a 10-min exposure to 300 mM citric acid (CA) and 0.4 M NaCl in ME medium (pH 3.5). Dually phosphorylated forms of Hog1p were detected using anti-phospho-p38. To check that equal amounts of protein were loaded in the lanes, Hog1p was detected using a polyclonal anti-C-terminal Hog1p antibody. A representative result is shown.