FIG. 6.
Myriocin inhibits phosphorylation of Slm1 and Slm2 during heat stress and steady-state growth conditions. GST-SLM2ΔPEFYIE (GBY116) and GST-SLM1ΔPNIYIQ (GBY118) were expressed in an slm1Δ slm2Δ strain. (A) Cells were grown at 25°C in selective synthetic medium for 4 h and protein expression was induced with 2% galactose. Thirty minutes before the shift from 25°C to 37°C, myriocin (2 μg/ml) was added to one set of cultures and solvent (methanol) to the other. Samples of the different cultures were taken at time points (t) just before the temperature shift and 90 min after the shift in temperature. GST fusion proteins were purified from cell extracts and subjected to SDS-PAGE and Western blotting with either an anti-phospho or an anti-GST antibody. (B) Cells were grown at either 25°C, 30°C, or 37°C in selective synthetic medium. Myriocin (2 μg/ml) was added 30 min before the cells were incubated with 2% galactose for 4 h. GST fusion proteins were purified from cell extracts and analyzed via Western immunoblotting with either an anti-phospho or an anti-GST antibody. Immunoreactive bands were quantified as described in Materials and Methods. A.U., arbitrary units.