FIG. 2.
Phosphorylation of C. heterostrophus BmHog1p MAPK induced by osmotic stress. (A) BmHog1p phosphorylation in the wild-type strain, the BmHOG1 disruption strain, and the E4509 strain. Mycelia of the strains HITO7711 (WT), DHOG101 (BmHOG1Δ), and E4509 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) Continuous activation of BmHog1p by NaCl in the wild-type strain. Mycelia of HITO7711 (WT) were incubated in CM with 0.8 M NaCl for 10, 30, and 60 min. Prepared mycelia were incubated in CM without NaCl as a control (indicated by C). The procedures for Western blot analysis were the same as those described above.