Visualizing Crz1-mCherry Localization

XW252 (26 (link), 27 (link)) was grown overnight for 16 hours in YPD medium at 30°C shaking and subsequently diluted to an OD₆₀₀ ~0.1 in fresh YPD medium. The cells were cultivated grown at 25°C to OD₆₀₀ ~ 0.5. Phenothiazine or DMSO was added to the culture and immediately placed in a water bath at the appropriate temperature for 15 minutes. Samples were fixed in 4% formaldehyde for 15 minutes, harvested by centrifugation and re-suspended in 50 μL of 40 mM potassium phosphate/1.2 M sorbitol buffer. Images were captured using Nikon ES80 epi-fluorescence microscope visualized with a CoolSnap CCD camera and NIS-Elements Software. Quantitative analysis was performed by visually inspecting images for co-localization of Crz1-mCherry with Nop1-GFP. The data represent the mean of at least two biological replicates for which at least 100 cells were counted. Error bars indicate standard deviation.