FIG. 2.

Phenotype and expression of alanine-scanning mutations in CSE629. (A) Cells lacking the YAP1 gene (Δyap1) were transformed with low-copy-number plasmids expressing the indicated forms of Yap1p or the vector only (pRS316). Transformants were grown to an A₆₀₀ of 1, and spots of 1,000 cells were placed on YPD containing diamide or H₂O₂. Each oxidant was present in a concentration gradient, as indicated by the bar at the top of the figure. (B) Δyap1 cells expressing the indicated forms of Yap1p were grown in the absence of oxidants (U) or challenged for 1.5 h with diamide (D) or H₂O₂ (H). Protein extracts were prepared, and either 50 μg (wild-type Yap1p) or 100 μg (mutants) of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel was then transferred to nitrocellulose and probed with a rabbit anti-Yap1p antiserum (24). Bound antibody was detected by using goat anti-rabbit antibody and chemiluminescence (Pierce).