Fig 3. CBF-1 rhythmically associates with the C-box of the frq promoter.

(A) EMSA assays showing GST-CBF-1 specifically binds to the C-box. A lower concentration of protein was utilized when protein was incubated with specific and non-specific probe. (B) Western blot analyses showing that polyclonal antibody specifically recognizes CBF-1 protein in the wild-type strain but not in the cbf-1^KO strain. The arrow indicates the CBF-1 protein band detected by CBF-1 polyclonal antibody. WT, wild-type; MW, molecular weight. (C) CBF-1 rhythmically binds to the C-box of frq promoter. Samples were grown for the indicated hours in DD prior to harvest for ChIP. (D) The levels of CBF-1 protein are consistent across circadian time points. (E) Rhythmic binding of WC-2 to the C-box is constantly high in the frq⁹ mutant. (F) Rhythmic binding of CBF-1 to the C-box is disrupted in wc-1, wc-2, and frq⁹ mutants. (G) The levels of CBF-1 protein were not altered in wc-1, wc-2, or frq⁹ mutants. Significance was assessed by using a two-tailed t-test. Data are means ± standard deviations (S.D.) (n = 3; **P<0.01 and ***P<0.001).