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. 2014 May 3;42(11):7436–7449. doi: 10.1093/nar/gku329

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© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — A Bat1 derived transcriptional activator (acBat1) is functional in a human cell reporter assay. (A) Schematic drawing showing the domain composition of acBat1. NLSs (yellow bars), a 3xFLAG tag (red crescent line) and a VP64 AD (green triangle) were fused onto Bat1 (purple) via flexible linkers (orange). This was introduced into HEK293T cells via transfection alongside a DNA reporter (grey) bearing BE _Bat1 (purple) upstream of a dsEGFP coding sequence (green). Transcriptional activation of the reporter (green arrow) follows binding to BE _Bat1, leading to production of dsEGFP protein (green star). acBat1 is detected via the 3xFLAG epitope with use of an Alexa Fluor 594 labelled secondary antibody. (B) Alexa Fluor 594, dsEGFP and DAPI fluorescence are shown for transfected cells. acBat1 is compared to derivatives lacking AD (acBat1ΔAD) or NLSs (acBat1ΔNLSs) and to a dTALE created with the same NLSs and AD and with the same core repeat number and RVD composition as Bat1 (dTALE_Bat1mimic). The scale bar indicates 10 μm. (C) FACS analysis was used to quantify dsEGFP fluorescence for transfected cells expressing acBat1, ΔAD derivative or dTALE_Bat1mimic as well as cells transfected with the reporter only. dsEGFP values are shown for the whole population (curves) as well as boxplots showing fold changes in fluorescence intensity compared to the reporter control. Boxplot whiskers represent the 2.5% and 97.5% data limits. Median values are written next to or inside each box plot and shown graphically with thick black lines.

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