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. 2017 Dec 18;7:17742. doi: 10.1038/s41598-017-18009-2

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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EsCLOCK and EsCYCLE/BMAL dimerize and activate transcription from the E-Box in vitro. (A) Co-immunoprecipitation of an epitope-tagged versions of EsCLK-V5 and MYC-EsCYC/BMAL co-expressed in HEK293 cells. Two experiments (Exp.) are reported showing that precipitates are enriched for EsClock-V5. Membranes were probed with anti-MYC antibody to visualize pulldown efficiencies. For presentation purposes western blot images have been cropped (full-length blots are presented in Supplementary Figure S8A-C). (B,C) EsCLK and EsCYC/BMAL luciferase assay. EsCLK and EsCYC/BMAL - only as a heterodimer - activate the transcription of an E-box luciferase reporter in S2R + and HEK 293 cells, respectively. Cells were transfected with indicated constructs. Negative control set as 1. Data are represented as mean ± SD (n = 3 independent transfections). (D) Identification of conserved domains responsible for the transactivation activity of the EsCLK:EsCYC/BMAL by luciferase assay and their selective deletion. Data are represented as mean ± SD (n = 3 independent transfections). See Supplementary Figure 7 for a schematic representation of the constructs generated. (E,F) Interactions between E. superba’s positive clock elements with those of D. melanogaster and M. musculus evaluated by luciferase assay in S2R + and HEK 293 cells, respectively. Negative control set as 1. Data are represented as mean ± SD (n = 3 independent transfections). Student’s t-test Bonferroni-corrected p-values for all the experimental comparisons discussed were presented in Supplementary Table 3. Statistical significance of the most relevant comparisons were shown as *p < 0.05, **p < 0.01, and ***p < 0.005.