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. 2014 May 23;32(6):1515–1526. doi: 10.1002/stem.1695

Figure 6.

Figure 6

Sox17 activates Cer1. (A–F): Mapping the Sox17 transactivation domain. (A) Schematic representation of the Sox17 deletion mutants. Domain of unknown function 3547 (Pfam 24.0) designates the conserved C-terminal region of F group Sox proteins. (B): Western blot analysis of the constructs in 293T cells. (C): Sox-dependent reporter gene activity (SOP-FLASH) in 293T cells, in the presence of cotransfected Sox17 expression vectors. Deletion of the N terminus (C1) increases the transcriptional activity. Deletions of the conserved C-terminal domain (N3, N4) attenuate transactivation. (D): Schematic representation of the GAL4DBD-Sox17 fusion proteins. (E): Western blot analysis of the constructs in 293T cells, using antibody to Gal4. (F): Reporter gene activity (5xGal4-luc) in 293T cells, induced by GAL4-Sox17 vectors. Deletion of the Sox17 C-terminal domain (GAL4 129–299) cripples transactivation. Other constructs showed activity equal to or greater than that of GAL4-VP16. (G, H): Doxycycline-dependence of the Sox17 vectors, measured in AB2.2 cells by flow cytometry (G), Western blotting (H, above), and transactivation of SOP (H, below). (I): Wild-type Sox17 and the C1 truncation both induce endogenous Cer1. n ≥ 3; *, p < .05 versus control embryonic stem cells. (J): The multimerized Sox17 site at −657 of the Cer1 locus mediates Dox-dependent, sequence-specific trans-activation. n = 6; *, p < .01 versus the absence of Dox.