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. Author manuscript; available in PMC: 2008 Jun 19.
Published in final edited form as: Curr Biol. 2007 Jun 7;17(12):1082–1089. doi: 10.1016/j.cub.2007.05.039

Figure 4. CWO Specifically Represses and Binds Clock Gene Promoters.

Figure 4

(A, B) S2 cells were cotransfected with reporter plasmids (1 μg except 0.1 μg for vri-mluc) and the increasing amounts (0.25 μg and 1 μg) of expression vector for FLAG-tagged CWO (AcF/CWO) in the presence (A) or absence (B) of expression vector for V5-tagged CLK (AcV5/CLK; 10 ng for per-luc, 0.25 ng for tim-luc and pdp1-mluc, 1 ng for vri-mluc). (C) S2 cells were cotransfected with reporter plasmids (1 μg) and the expression vector for FLAG-tagged CWO or VP16 activation domain-fused CWO (1 μg). Activation fold was calculated by normalizing values to luciferase activity in the presence of reporter plasmid, which was set to 1, while repression fold by inversely normalizing them. For (A-C), N experiments = 3, and standard deviations are depicted by error bars. (D) Electrophoretic mobility shift assay for CWO binding to E boxes (CACGTG). Increasing amount (10- and 50- fold molar excess) of unlabeled competitors containing wild-type (E-box) or mutant E-box (mE-box) or 2 μg of anti-GST or anti-FLAG was preincubated with GST-fusion proteins prior to the addition of labeled probe. C1, shift by GST-CWO bHLH:DNA complex; C2, supershift by GST-bHLH:DNA:anti-GST antibody complex.