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. 2018 May 24;8:8078. doi: 10.1038/s41598-018-26281-z

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Expression of RT variants in eukaryotic cells. (a–f) Western blotting of the lysates of HeLa cells transfected with vector pVax1 (lane 1), and pVax-based plasmids expressing RTwt (lane 2 a,c,e), RTwt-opt (lane 3 a,c,e), RTwt-opt-in (lane 4 a,c,e), RT1.14 (lane 2 b,d,f), RT1.14-opt (lane 3 b,d,f), and RT1.14-opt-in (lane 4 b,d,f). Blots of RTwt and RT1.14 variants were processed in parallel. Blots were stained with rabbit polyclonal anti-RT antibodies⁵⁹ (a–d) and then stripped and re-stained with monoclonal anti-actin antibodies. (e,f) Positions of the relevant molecular mass markers are given to the right in kDa. Arrows point at the p66 and p51 RT subunits. Panels (a,b) represent results of a 0.5 min exposure of X-ray film with a blot, while panels (c,d) represent results of a 10 min exposure. Full-length blots are presented in Supplementary Fig. 2. (g) The average amount of RT protein expressed per HeLa cell transfected with RT variant genes. **p < 0.01 (as compared to non-optimized RT variants) using the Mann–Whitney U-tests. The graph represents the results of three independent runs, each done in duplicate, and the error bars represent the SD.