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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 4. — Abundance of dsRNA alters the requirement of the E3L ZBD for PKR inhibition in mammalian cells. (A) Human HeLa PKR^kd cells were cotransfected with expression vectors for luciferase, an empty vector or knockdown-resistant human PKR (100 ng), and C-terminally HA-tagged variola E3L-HA or E3L-Δ80-HA (400 ng). After 40 h, cells were harvested, lysed, and extracts were assayed for luciferase activity. Luciferase activity was normalized to the transfections containing PKR but lacking E3L. Error bars indicate the standard deviation for three independent transfections. (B) Analysis of luciferase activity following cotransfection of PKR^kd cells with poly(I:C) (500 ng) and the expression vectors for luciferase, vector or PKR (100 ng), and E3L-HA or E3L-Δ80-HA (400 ng) as described in panel A. (C) Immunoblot analyses of WCEs from cells transfected as described in panels A and B with or without poly(I:C) and the indicated expression vectors for PKR, E3L, or E3L-Δ80. Blots were probed with antibodies specific for phosphorylated Ser51 in eIF2α (top panel), polyclonal antiserum against human eIF2α (second panel), monoclonal human PKR antibody (third panel), and monoclonal anti-HA antibody to detect HA-tagged E3L (fourth panel). Note that E3L-HA resolves as two species on SDS-PAGE (see text). (*) E3L-Δ80 comigrates with a nonspecific protein that cross-reacts with the anti-HA antibody. (Bottom panel) Relative level of eIF2α-P to total eIF2α was determined for three independent experiments using quantitative densitometry and Image J software (NIH) and normalized to the vector transformant.