Expression of HA-Ppt1p as a function of cell growth. (A) An overnight culture of WHT4-1 cells (OD600 nm 5~10) was used to inoculate fresh medium to OD600 nm 0.2 and the culture grown at 30°C. At the indicated times samples were prepared and subjected to immunoblotting using anti-HA antibody. The lower panel shows the bottom half of the same blot probed with anti-Cdc28p antibody. This experiment was performed four times independently with similar results. WT; wild type. (B) The growth curve was plotted based on direct cell counting using a hemocytometer. The relative amount of HA-Ppt1p was obtained by taking the ratio of the band intensity of HA-Ppt1p to Cdc28p. (C) Total RNA was isolated at the indicated time after the inoculation of an overnight culture of W303 cells (OD600 nm 5~10) to OD600 nm 0.2. Twenty micrograms of RNA per sample were resolved and subjected to Northern analysis with a 32P-labeled PPT1 cDNA probe. The lower panel shows the same blot probed for yeast actin mRNA. This experiment was repeated four independent times for both W303 and WHT4-1 cells with similar results.