Figure 3.

FRQ, WC-1, and WC-2 are hypophosphorylated in the ck-1a^L strain, a knock-in mutant carrying the dbt^L mutation. (A) Graphic diagram depicting the procedures to create the homokaryotic ck-1a^L strain by homologous recombination (see Materials and Methods for details). The asterisk in the ck-1a ORF indicates the location of the dbt^L mutation. (B) Southern blot analysis showing the insertion of hph in the endogenous ck-1a locus. The genomic DNA was digested by SphI and the membrane was probed with a ck-1a-specific probe. (C) Western blot analyses showing that hypophosphorylation of FRQ, WC-1, and WC-2 in the ck-1a^L strain. Cultures were grown in LL. The arrows indicate the extensively phosphorylated FRQ or WC species absent in the mutants. The numbers on the right indicate the ratio of acrylamide/bisacrylamide used in the SDS-PAGE. Note that for WC-2, the regular SDS-PAGE (37.5:1) failed to resolve different WC-2-phosphorylated forms. (D) Western blot analyses showing the stability of FRQ, WC-1, and WC-2 after the addition of CHX (10 μg/mL) in the wild-type and ck-1a^L strains. Cultures were grown in LL. (E) Densitometric analysis from four independent experiments showing the degradation of FRQ in the wild-type and ck-1a^L strains after the addition of CHX.