Figure 4.

Reduction of CLOCK and BMAL1 interaction and nuclear localization of CLOCK by CLK8. A, effect of CLK8 on the interaction of CLOCK and BMAL1 were evaluated by co-immunoprecipitation. Anti-FLAG affinity gel was used to precipitate FLAG-tagged CLOCK together or FLAG-tagged CLOCK-F80A,K220A mutant with BMAL1. Western blot analysis using anti-BMAL1 and anti-FLAG antibodies was performed to compare the association of CLOCK and the CLOCK-F80A,K220A mutant with BMAL1 in samples with different concentrations of CLK8. Quantification of the Western blotting is shown in the right panel. The vertical axis, which indicates the amount of BMAL1 (normalized by CLOCK) in each sample, was calculated relative to the DMSO control. B, unsynchronized U2OS cells were treated with 20 μm CLK8. Control cells were treated with 0.5% DMSO. Two days later, cells were fractionated and examined by Western blot analysis, where CLOCK, NPAS2, PER2, BMAL1, and CRY1 were normalized by α-tubulin or histone-H3. Quantifications are shown on the right. Data are mean ± S.E.; n = 5 independent experiments. The Western blotting data are representative of five independent experiments. *, p value <0.05; **, p value <0.01.