Localization of Stress Response Proteins

Localization of Ssk1-GFP and Hog1-GFP were performed as described [8 (link)]. Overnight cultures of cells expressing Ssk1-GFP and Hog1-GFP were diluted in the morning to an OD₆₀₀ ~ 0.1 in HC-Leu with 1μg/mL of DAPI and grown for three doubling times to early log phase (OD₆₀₀ ~ 0.8–1.0). Aliquots of cell were left in HC-Leu or washed once in HC-Leu without 2% glucose (HC-Leu-Glc) and incubated for 30 min in HC-Leu-Glc or submitted to osmotic stress with 0.4 M NaCl (HC+NaCl) for 5 min. GFP and DAPI signals in living yeast cells were viewed using a Nikon fluorescence microscope (model E800) equipped with a Plan-Apo 100x/1.4 oil objective and a Photometrics Coolsnap HQ camera. Images were analyzed using Metamorph software (Universal Imaging Corporation).