Cell growth and treatment with αF and NaCl
This protocol is extracted from research article:
Engineering allosteric regulation in protein kinases
Sci Signal, Nov 6, 2018; DOI: 10.1126/scisignal.aar3250

All cells were grown in synthetic complete medium with dextrose (SDC). Three single colonies from each Kss1 or Hog1 strain were inoculated in 1 ml of SDC in 2-ml 96-well deep-well plates and serially diluted 1:5 three times. Plates were incubated overnight at 30°C. In the morning, cells from the row that had been diluted 1:25 were typically found to have OD600 (optical density at 600 nm) ~ 0.5. These cells were diluted 1:5 in four rows of a 96-well U-bottom microtiter plate in a total volume of 180 μl and incubated for 1 hour at 30°C. For Kss1 strains, in each row, cells were treated with different concentrations of αF: 0, 0.01, 0.1, and 1 μM (10× stocks of αF were prepared, and 20 μl was added to 180 μl cells). Treated cells were incubated for an additional 4 hours at 30°C before translation was stopped by addition of cycloheximide (50 μg/ml). Cells were incubated for an additional hour at 30°C to allow time for fluorophores to mature. For Hog1 strains, 0 or 0.5 M NaCl was added for 2 hours before cycloheximide arrest. For experiments with estradiol, everything is the same except that all media contained 20 nM estradiol for the duration of the overnight growth and throughout the experiment.

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