FIG. 5.

The cellular level of Spc1 MAPK, but not Wis1 MAPKK, is reduced in the cdc37 mutant. (A) Wild-type (CA178) and cdc37-681 (CA1450) strains, in which chromosomal wis1⁺ is tagged with the sequence encoding the myc epitope, were grown to the mid-log phase at 30°C in YES medium, and their cell lysate was subjected to immunoblotting with anti-Spc1 and anti-myc antibodies to examine the protein levels of Spc1 and Wis1myc, respectively. (B) Wild-type (PR109) and cdc37-681 (CA1388) cells exponentially growing in YES medium at 30°C were harvested and subjected to a Northern hybridization analysis to quantify the spc1⁺ mRNA levels. The results were quantified and normalized by the leu1 mRNA levels using the Storm system (Molecular Dynamics). (C) The cellular levels of Spc1 MAPK in wild-type (PR109), cdc37-681 (CA1388) and swo1-26 (CA1496) strains were examined by anti-Spc1 immunoblotting as described for panel A. The protein level of Spc1 is not affected by the Hsp90 mutation, swo1-26. Immunoblotting with anti-tubulin antibodies (lower panel) showed that the amounts of the cell lysate loaded to each lane were comparable.