FIG. 3.

Half-time determinations for c-myc proteins in yeast cells. W303-1A or BJ2168 cells expressing the protein of interest were grown to mid-log phase in galactose selective medium. Glucose was added to 5% to repress expression of the c-myc derivatives, and 10-ml aliquots were removed from the cultures at various times thereafter. Cell extracts were made as described for Fig. 1B. The volumes of cells for each extract were adjusted if necessary after the OD₆₀₀ was determined. A 30-μl volume of each sample was subjected to SDS-PAGE (7.5% polyacrylamide), and Western blotting was performed as described for Fig. 1B, except where indicated. The intensity of each band was determined with GelPro image analysis software. The amount of protein present at time zero was set to 100%, and the percentages remaining were plotted on a logarithmic scale against time to calculate the half-time. (A) Half-time determination for intact full-length c-myc. The half-time was measured for two different BJ2168 transformants. A representative Western blot is shown, and the values used to calculate the half-time are the means from two experiments. The c-myc protein is undetectable in W303-1A cells, so the half-time could not be measured in this strain. The open arrow indicates a nonspecific band detected by the antibody. (B) Half-time determinations for full-length c-myc lacking the two myc boxes. The half-time was measured in duplicate for transformants of both W303-1A and BJ2168. Representative Western blots are shown, and the values used to calculate the half-time are the means from two experiments. (C) Half-time determination of β-galactosidase. The primary antibody used was against β-galactosidase (1:500 in 1% milk–phosphate-buffered saline–Tween), and the secondary antibody was sheep anti-mouse IgG-HRP conjugate. The measured values for the intensity of the β-galactosidase bands were corrected with an internal loading control (results not shown).