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. 2014 Feb;20(2):214–227. doi: 10.1261/rna.042341.113

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Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported), as described at http://creativecommons.org/licenses/by-nc/3.0/.

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FIGURE 2. — Genetic evidence that the Z-DNA binding domain of E3L is essential for PKR inhibition in yeast. (A) Domain architecture of variola E3L and human PKR. (ZBD) Z-DNA binding domain; (dsRBD) dsRNA-binding domain. (B) Transformants of yeast strains J110 (−PKR) and H2544 (+PKR) bearing empty vector pEMBLyex4 or plasmids that express the indicated E3L protein under the control of a GAL–CYC1 promoter were grown to saturation, and 5 µL of serial dilutions (of OD₆₀₀ = 1.0, 0.1, 0.01, and 0.001) was spotted on SCGal (10% galactose) medium and incubated for 3 d at 30°C. (C) Immunoblot analysis of E3L and PKR. WCEs were prepared from the yeast transformants described in B, and 5 µg of total protein extracts was subjected to SDS-PAGE followed by immunoblot analysis using monoclonal antibodies directed against PKR and the HA-epitope to detect N-terminally HA-tagged E3L or polyclonal antisera against yeast eIF2α.