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. 2018 Sep 20;46(21):11539–11552. doi: 10.1093/nar/gky851

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© The Author(s) 2018. 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/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — CBP80 cooperates with the functions of Rev on Gag synthesis from the unspliced mRNA. (A) HeLa cells were transfected with 1 μg pNL4.3-wt together with 1 μg pCMV-myc-CBP80 or 1 μg pCMV-myc-eIF4E. At 24 hpt, the interaction between the unspliced mRNA and the myc- tagged protein was analyzed by the ISH-PLA protocol described in materials and methods. Red dots indicate the interactions between the unspliced mRNA and the corresponding myc-tagged protein. Scale bar 10 μm. A quantification of the dots/cell in each condition is presented on the right (****P < 0.0001, Mann–Whitney test). (B) HeLa cells were transfected with 0.3 μg of pNL4.3R or pNL4.3R ΔRev proviruses together with 1 μg of pCMV-myc-CBP80 or pCMV-myc-eIF4E (pCMV-myc-d2EGFP was used as control). At 24 hpt, cell extracts were prepared for Gag-Renilla activity measurement and for cytoplasmic RNA extraction and RT-qPCR analyzes. Results for Gag synthesis (left panel), cytoplasmic unspliced mRNA (middle panel) and translational efficiency (right panel) were normalized to the wild type provirus (arbitrary set to 100%) and presented as the mean ± SD of three independent experiments (*P < 0.05; ***P < 0.001 and NS; non-significant, t-test). (C) HeLa cells were transfected with 0.3 μg of pNL4.3R or pNL4.3R ΔRev proviruses together with 1 μg of pCMV-myc-CBP80 (pCMV-myc-d2EGFP was used as control) as described in materials and methods. At 24 hpt, cell extracts were prepared for Gag-Renilla activity measurement and for cytoplasmic RNA extraction and RT-qPCR analyzes. Results for Gag synthesis (left panel), cytoplasmic unspliced mRNA (middle panel) and translational efficiency (right panel) were normalized to the wild type provirus (arbitrary set to 100%) and presented as the mean ± SD of three independent experiments. (*P < 0.05; **P < 0.01 and NS; non-significant, t-test). (D) HeLa cells were transfected with 1 μg pCDNA-Flag-Rev and 1 μg pCDNA-V5-CBP80. At 24 hpt, the interaction between Flag-Rev and V5-CBP80 was analyzed by PLA. Red dots indicate interactions between both proteins. Scale bar 10 μm.