Fig 10. Hog1 pathway influences the duration of hyperosmotic stress induced sumoylation and Cyc8^4KtoR inclusions.

(A) His₆-FLAG-SMT3 cells with the HOG1 or hog1Δ allele were subjected to a time course of hyperosmotic stress (1.2M sorbitol). Changes in sumoylation patterns were examined by western analysis using an anti-FLAG antibody. (B) His₆-FLAG-SMT3 cells expressing either wild-type Cyc8 or mutant Cyc8^4KtoR were subjected to a time course of hyperosmotic stress (1.2M sorbitol). Phosphorylated Hog1 (top panel), wild-type Cyc8 and mutant Cyc8^4KtoR levels (middle panel), and sumoylated wild-type Cyc8 and mutant Cyc8^4KtoR levels (bottom panel) were examined by western analysis using an anti-phosphorylated Hog1 antibody and the HSV antibody that detects Cyc8-3HSV. (C) SIM images of HOG1 or hog1Δ cells under a time course of hyperosmotic stress. Wild-type Cyc8 and mutant Cyc8^4KtoR are tagged with GFP, and the cell wall is stained with Calcofluor White. Quantitation of results is shown. The presence of inclusions was analyzed by counting the cells with at least one visible inclusions. At least 200 cells in at least 5 fields were analyzed for each time point. Data in the graph represent the average of 3 independent experiments. Error bars represent the standard deviation.