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. 2017 Aug 22;114(36):E7516–E7525. doi: 10.1073/pnas.1702014114

Fig. 4.

Fig. 4.

Several domains on dpCLK and dpBMAL1 contribute to transcriptional repression by dpCRY2 in S2 cells. (A, Upper) DpCRY2 does not repress on the BMAL1 G and ΔCter mutant TAD domains. A UAS luciferase reporter (UAS_Luc; 10 ng) was used in the presence (+) or absence (−) of Gal4DBD, Gal4DBD_G+TAD, Gal4DBD_G+ΔCter, Gal4DBD_G, and Gal4DBD_TAD expression plasmids (5 ng each), and increasing doses of dpCRY2 (amounts are given in nanograms). Firefly luciferase activity was computed relative to renilla luciferase activity. Each value is the mean ± SEM of three replicates. (Lower) Western blots of V5-tagged dpCRY2 and Drosophila β-actin protein expression levels. (B) DpCRY2 inhibits dpCLK:dpBMAL1- and dpCLK:dpBMAL1ΔCter-mediated transcription. The monarch per E box luciferase reporter (dpPerEp_Luc; 10 ng) was used in the presence (+) or absence (−) of dpCLK, dpBMAL1, dpBMAL1ΔCter, dpBMAL1ΔTAD, and dpCYC-like expression plasmids (5 ng each) and increasing doses of dpCRY2 (amounts are given in nanograms). Quantification of luciferase activity, values, and Western blot analysis are shown as in A. (C) DpCRY2 dose-dependent inhibition of dpCLK:dpBMAL1 mutants fused to the VP16 transactivation domain in their N termini. The monarch per E box luciferase reporter (dpPerEp_Luc; 10 ng) was used in the presence (+) or absence (−) of dpCLK, dpBMAL1, VP16-dpBMAL1, VP16-dpBMAL1ΔCter, VP16-dpBMAL1ΔTAD, and VP16–dpCYC-like expression plasmids (5 ng each) and increasing doses of dpCRY2 (amounts are given in nanograms). Quantification of luciferase activity, values, and Western blot analysis are depicted as in A. One-way ANOVAs for dose-dependent repression by dpCRY2 on each BMAL1 variant: P < 0.0001 to P < 0.005. (D) DpCLK is required for VP16-dpBMAL1–mediated transcription. The monarch per E box luciferase reporter (dpPerEp_Luc; 10 ng) was used in the presence (+) or absence (−) of dpCLK and VP16-dpBMAL1 expression plasmids (5 ng each). Quantification of luciferase activity and values are depicted as in A. (E) DpCRY2 does not repress the VP16 activation domain. A UAS luciferase reporter (UAS_Luc; 10 ng) was used in the presence (+) or absence (−) of Gal4DBD and Gal4DBD-VP16 expression plasmids (5 ng each) and increasing doses of dpCRY2 (amounts are given in nanograms). Quantification of luciferase activity, values, and Western blot analysis are depicted as in A. P = 0.79 (one-way ANOVA).