FIG. 4.
Phosphorylation of C. heterostrophus BmHog1p MAPK in the wild-type strain and the dic1 mutants E4504 and N9006. (A) BmHog1p phosphorylation by osmotic stress. Mycelia of the strains HITO7711 (WT), E4504 and N9006 were used for analysis. Prepared mycelia of the strain tested were incubated in CM with or without 0.4 and 0.8 M KCl for 10 min. The cells were harvested, and total protein extracts were prepared as described in Materials and Methods. Protein samples (50 μg) were subjected to SDS-PAGE and blotted onto nitrocellulose membranes for Western blot analysis. Phosphorylated BmHog1p was detected using anti-dually phosphorylated p38 antibody (indicated by anti-TGYp). The total amount of BmHog1p was measured using anti-Hog1 C terminus antibody (indicated by anti-Hog1). (B) BmHog1p phosphorylation by iprodione in the wild-type strain and the dic1-deficient strains. Mycelia of HITO7711 (WT), E4504, and N9006 were used for analysis. Prepared mycelia were incubated in CM with 10 or 100 μg/ml iprodione for 10 min. Prepared mycelia of the strain tested were incubated in CM without iprodione as a control (indicated by C). The procedures for Western blot analysis were the same as those described above.