- + - + - + KCl Pbs2 Pbs2AA Pbs2DD

Supplementary Figure 1. Phosphotyrosine analysis of Hog1. Western blot analysis of whole cell extracts isolated from PBS2-HM (JC112), PBS2AA-HM (JC126) and PBS2DD-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).

Hog1-TyrP*

Hog1