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. 2003 Apr 24;100(10):5914–5919. doi: 10.1073/pnas.1030057100

Figure 4.

Figure 4

The C box is rhythmically bound by the WC-1/WC-2 complex. To determine whether FRQ acts by blocking binding of the WC-1/WC-2 complex to the C box or by blocking activation by a constitutively bound complex, C-box binding by WC-1 and WC-2 was determined as a function of CT. (A) EMSA using the C-box probe and nuclear protein extracts from cultures harvested across a circadian day reveals changes in the amount of complex bound but not in its apparent size. The arrow indicates the WC-1/WC-2 complex. (B) Western blot analysis of FRQ, WC-1, and WC-2 in the nuclear protein extracts used in A confirm appropriate rhythmicities. (C) Densitometry of the C-box-bound complex and FRQ shows that the amount of complex bound to the C box changes over the course of a circadian day, peaking ≈6 h before the peak in FRQ protein. The lowest value in each group was set to 1. The experiment was repeated multiple times with similar results; representative data are shown.