FIG. 8.

Levels of Nrg1 and Sfl1 increase in the presence of glucose. (A) Wild-type cells were grown in a synthetic medium containing 2% glucose (R) or 2% glycerol-ethanol (D) as carbon sources, and total RNA was prepared for Northern blot analysis. The yeast actin gene (ACT1) was used as an internal control. (B) A plasmid containing SFL1p-lacZ was transformed into wild-type cells, and β-galactosidase activity was determined under the same conditions from three independent colonies. (C) Nrg1-HA and Sfl1-HA were expressed from their own promoters. Cells were grown in synthetic medium with 2% glucose or 2% glycerol-ethanol to mid-log phase, and total cellular proteins were prepared. Western blot analysis was performed to determine the levels of Sfl1-HA and Nrg1-HA from 50 μg of protein extract. The same membranes were probed with an α-actin monoclonal antibody. (D) Cells were grown to mid-log phase in synthetic medium with 2% glycerol-ethanol and shifted to synthetic medium with 4% glucose until they reached an OD₆₀₀ of 4.0. Total RNA was prepared at the indicated time points for Northern blot analysis. The blot was hybridized with an NRG1 probe and then stripped and rehybridized with STA1 and ACT1 probes.