Experimental Investigation of Protein Depletion Below Normal Endogenous Levels for Dbf4, Cdc6 and Cdt1. (A) Asynchronous cultures of GAL1-CDC6 (DY-139), GAL1-CDT1 (DY-140), GAL1-DBF4 (DY-255) and their wild-type counterparts DY-142, DY-143 and DY-256, respectively, were grown to 106 cells/ml in galactose (GAL) medium, washed and resuspended in glucose (GLU) medium. Whole-cell extracts were prepared from culture aliquots taken prior and post shift from galactose to glucose with indicated time points corresponding to time in glucose medium. HA-tagged Cdc6 and HA-tagged Cdt1 were detected using an anti-HA antibody (Sigma) and a fluorescent secondary antibody (Invitrogen). Ponceau S staining of the region detected by the blot to judge loading of whole-cell extracts is also shown. (B) FACS analysis of culture aliquots from either asynchronous (Async) cultures, or at the indicated times after cell resuspension in glucose medium.