FIG. 8.

Activity and phosphorylation of Hog1 double mutants is further enhanced by osmotic stress in hog1Δ cells but not in pbs2Δ cells. hog1Δ or pbs2Δ cells expressing the indicated Hog1 molecules were grown in the absence of methionine for 2 h. Half of each culture was subjected for 10 min to 1 M NaCl. (A) The upper panels show two exposures of the same autoradiogram of a kinase assay performed with the various Hog1 proteins immunoprecipitated from cells treated (+) or untreated (−) with 1 M NaCl. The lower panel shows a Western blot of the immunoprecipitated lysates. (B) Western blot analysis of whole-cell lysates prepared from the indicated strains. The upper panel shows the level of dually phosphorylated Hog1 when anti-phospho-p38 antibodies are used. The lower panel shows the total Hog1 protein levels when anti-HA antibodies are used.