Fig. 5.

Cip1 is phosphorylated by Hog1 kinase directly under osmotic stress. a Isogenic WT strains bearing empty vector, GAL1-CIP1, GAL1-cip1-3TA, or GAL1-cip1-3TE plasmid were spotted in 10-fold diluted equal number of yeast on 2% glucose and 2% galactose plates. b Recombinant GST-Sic1, GST-Cip1, and GST-Cip1-3TA were employed as substrates for in vitro kinase assay. GST-Hog1 was activated by incubation with GST-Pbs2^EE for 30 min at 30 °C. Activated Hog1 was added to the substrate and incubated at 30 °C for 30 min in the presence of ³²P-ATP. Proteins were resolved by SDS–PAGE and stained with Coomassie blue (bottom), and phosphorylated proteins were detected by autoradiography (top). Sic1 was included as a positive control; SB203580 was used to selectively inactivate Hog1. c Cip1 phosphorylations in WT cells, WT cells treated with 0.5 M KCl, and hog1 cells treated with 0.5 M KCl were detected by phosphor-specific antibodies, pT65 and pT73, and total Cip1 was served as a loading control. The mobility shift of phosphorylated Cip1 was shown in low percentage gel