Fig 4.

Mpk1 phosphorylation is induced in wild-type cells in response to thermal stress and the anti-Mpk1 phospho-antibody is specific for phosphorylation of Mpk1. (A) KN99α cells harboring the Mpk1 Flag-tagged construct were grown overnight in YPD medium at 24°C and then split the next morning into two aliquots. To one aliquot, an equal volume of YPD medium at room temperature was added, and cells were grown for an additional 15, 30, 45, and 60 min at 24°C. To the second aliquot, an equal volume of YPD medium prewarmed to 55°C was added, and the cells were heat shocked for the same number of minutes at 39°C. Western blots containing 50 μg of total protein from cell lysates were analyzed by probing sequentially with anti-Mpk1 phospho-antibody and then anti-Flag antibody after the membrane was stripped. (B) The Mpk1 Flag-tagged and mpk1Δ strains were treated as described for panel A, but heat shocked for 1 h only. Blots were probed sequentially with anti-Mpk1 phospho-antibody, anti-Flag antibody, and finally anti-β actin antibody for a loading control after the membrane was stripped.