(A) mtl2Δ and wsc1Δ cells do not show the vic phenotype. Wild-type (YS64), rgf1Δ (VT14), pmk1Δ (MS192), mtl2Δ (YS1220), and wsc1Δ (SC136) were dropped onto the plates as indicated and then incubated for 4 days at 28°C. (B) Strains MI200 (pmk1-HA6H, control), SC19 (pmk1-HA6H, mtl2Δ), and SC141 (pmk1-HA6H, wsc1Δ) were grown in YES medium to mid log phase and treated with 1 μg/mL of Csp and grown for 60 min or treated with 15 mmol/L caffeine and grown for 2 h. Pmk1-HA6H was purified by affinity chromatography under native conditions from samples with or without treatment. Activated and total Pmk1p were detected by immunoblotting with anti-phospho-p42/44 or anti-HA antibodies, respectively. (C) The same experiments described in A, except that the cells were subjected to 0.6 mol/L KCl for 15 min or 0.5 mol/L NaCl for 30 min. Each treatment was repeated at least three times. Relative units comparing the induction-fold of wild type and the mutant cells in each individual experiment are shown below.