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. 2014 Aug;20(8):1238–1247. doi: 10.1261/rna.043141.113

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© 2014 Zhabokritsky et al.; 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 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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FIGURE 3. — PAP causes RRE-free mRNAs to accumulate on polysomes. (A) 293T cells were cotransfected with pMenv(-) proviral clone (5 μg) and pcPAP or pcDNA3 (2.5 μg). Cells were harvested 40 h post-transfection, and lysates were resolved by density gradient centrifugation and collected in 12 fractions. RNA was isolated from each fraction and levels of the three HIV-1 mRNA classes were visualized by Northern blotting following hybridization with the HIV-1-specific probe. The polysome profile was visualized prior to Northern blotting by ethidium bromide staining of the agarose gel. (B) The abundance of HIV-1 mRNAs in each size class was measured by quantifying the band intensity using a PhosphorImager and the ratio of RRE-free mRNAs (2 kb) to RRE-containing mRNAs (9 and 4 kb) was plotted. (C) The abundance of HIV-1 mRNAs on polysomes was plotted as a percentage of total HIV-1 RNA. Error bars in B and C represent means ± SE for three independent experiments.