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. 2014 Apr 20;42(10):6645–6658. doi: 10.1093/nar/gku304

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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 6. — Effect of TAP-p15 overexpression on HIV-1 expression. (A) Genome organization of HIV-1 NL4-3ΔRev. The start codon of the Rev gene was mutated from ATG to ACG. (B) HEK293T cells in a 6-well plate (70% confluent) were transfected with pcDNA3-GFP (1 μg), pNL4-3ΔRev (1 μg), and either pCI-neo (0.1 μg) (−) or pCI-FLAG-Rev (0.1 μg) (+). After 24 h, cells were collected and proteins from cell pellets were analyzed by SDS-PAGE and western blotting with rabbit anti-Gag^p55 antiserum. UT: untransfected cells were used as a control. (C) HEK293T cells in a 6-well plate (70% confluent) were transfected with pcDNA3-GFP (1 μg), pNL4-3ΔRev (1 μg), pCI-FLAG-Rev (0.1 or 1 μg), and either pcDNA5 (1 μg) (−) or pcDNA5-FLAG-TAP (0.6 μg) plus pcDNA5-FLAG-p15 (0.3 μg) (+). 0.9 μg of pCI-neo was added for 0.1 μg of pCI-FLAG-Rev to equalize the amount of plasmid DNAs. Supernatants and cells were collected after 24 h. RNA from cell pellets was subjected to semi-quantitative RT-PCR. PCR products were analyzed by electrophoresis in a 2% agarose gel. (D) Quantitation of the relative level of RNAs from three independent experiments performed as in (C). GFP mRNA was used for normalization. 0.1 μg of the pCI-FLAG-Rev sample (lane 1) was set to 1. Averages and standard deviations are shown. (E) Protein from the cell pellets in (C) was analyzed by SDS-PAGE and western blotting with rabbit anti-Gag^p55 antiserum or the anti-GFP antibody. UT: untransfected cells were used as a control. U2AF⁶⁵ was a loading control. (F) The filtrated media from (C) were immunoprecipitated with rabbit anti-Gag^p55 antiserum and detected by western blotting with the monoclonal anti-Gag^p24 antibody.