Figure 1.

Crz1p is dephosphorylated by calcineurin in vitro. HA-Crz1p was purified by immunoprecipitation from crz1Δ strains ASY472 (WT) and ASY475 (cnb1Δ) containing pAMS466 [YEp(HA–CRZ1)], incubated under different conditions as noted, and analyzed by PAGE and Western blotting (see Materials and Methods). (Lanes 1,6) HA–Crz1p incubated with buffer; (lanes 2,7) HA–Crz1p incubated in buffer with 20 mm CaCl₂, and calmodulin; (lanes 3,8) HA–Crz1p incubated in buffer with 20 mm CaCl₂, 83 units of calmodulin, and 0.8 unit of calcineurin, as described in Materials and Methods; (lanes 4,9) HA–Crz1p incubated in buffer with calcineurin and calmodulin plus 100 mm EGTA; (lanes 5,10) HA–Crz1p incubated in buffer with 20 mm CaCl₂ and calmodulin plus calcineurin that was preincubated for 15 min at 30°C with 100 μm cypermethrin.