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- + - + - + KCl

Pbs2. Pbs2 AA. Pbs2 DD. - + - + - + KCl. Hog1-TyrP. *. Hog1.

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- + - + - + KCl

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  1. Pbs2 Pbs2AA Pbs2DD - + - + - + KCl Hog1-TyrP * Hog1 Supplementary Figure 1. Phosphotyrosine analysis of Hog1. Western blot analysis of whole cell extracts isolated from PBS2-HM(JC112), PBS2AA-HM (JC126) andPBS2DD-HM(JC124) cells before and after treatment with 0.6 M KCl for 10 mins. Western blots were probed with an anti-phosphotyrosine antibody (4G10, Upstate). A cross-reactive band than runs below Hog1-TyrP is present in all samples (*). Due to problems with stripping blots of the 4G10 antibody, total levels of Hog1 protein was determined by running duplicate samples and probing the blot with an anti-Hog1 antibody which recognises both phosphorylated and unphosphorylated forms of Hog1 (Hog1).

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