FIG. 6.

War1p is reversibly phosphorylated during weak acid stress. (A) Exponentially growing YAK101 (WAR1-HA) cells were treated with 8 mM sorbate for the time periods indicated. War1p-HA and Pdr12p were immunodetected in cell extracts with monoclonal anti-HA antibody 12CA5 and polyclonal anti-Pdr12p antibodies, respectively. (B) YAK101 cells were stressed with 8 mM sorbate for 1 h in YPD or left unstressed. Protein extracts were incubated with phosphatase (CIP) for 1 h at 37°C, mock treated, or treated with CIP in the presence of orthophosphate as an inhibitor. (C) YAK101 cells were grown to an OD₆₀₀ of 1.0; then, 8 mM sorbate was added for the time periods indicated (sorbate). Sorbate was removed by spinning out cells and resuspending them in fresh prewarmed YPD medium (YPD). Samples were taken, and protein extracts were analyzed by immunoblotting with anti-HA antibody 12CA5 and anti-Pdr12p antibodies.