FIG. 2.

Slow-migrating Skn7 variant seen in oxidative stress-treated cultures is eliminated by phosphatase treatment and in a yap1Δ strain. TCA extracts prepared from JF1904 (skn7Δ) or JF2312 (skn7Δ yap1Δ) cultures carrying SKN7 or skn7-D427N expression plasmids and left unexposed (−) or exposed (+) to an oxidant (ox) or other (heat shock, wall, and osmotic) stress treatments were subjected to SDS-10% PAGE for 2.5 h at 200 V. Lambda phosphatase (Ph) was added where indicated to 100 U for 30 min at 30°C according to the manufacturer's recommendations. Representative experiments are shown. (A) Extracts were prepared from oxidant-treated and untreated cultures of the skn7Δ strain JF1904, carrying the SKN7 plasmid pXH1853. Phosphatase inhibitors (In) were added to all lanes except the lane labeled Ph. A dashed white line is added to the figure based on the migration of two untreated wild-type samples run as controls on the outside edges of the gel. (B) Extracts were prepared from oxidant-treated and untreated cultures of the skn7Δ strain JF1904 (left two lanes) and the skn7Δ yap1Δ strain JF2312 (right two lanes), each carrying the SKN7 plasmid pXH1853. (C) Extracts were prepared from oxidant-treated and untreated cultures of the skn7Δ strain JF1904, carrying the SKN7 plasmid pXH1853 (left two lanes) or the skn7-D427N plasmid pXH1858 (right two lanes). (D) Extracts were prepared from treated and untreated cultures of the skn7Δ strain JF1904, carrying the SKN7 plasmid pXH1853. The two lanes on the left are without or with oxidant treatment, while the two lanes on the right are without or with the indicated alternative stress treatments, including osmotic (0.4 M NaCl for 5 min), heat shock (39°C for 20 min), and wall stress (1 U/ml zymolyase for 30 min).