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. 2018 Jan 24;9(2):103. doi: 10.1038/s41419-017-0116-2

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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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Fig. 2 — a HeLa cells transfected with control plasmid (lanes 2, 4, 6, 8) or Flag-tagged N4 (lanes 3, 5, 7, 9) were mock-infected (lane 1) or infected with HSV-1/F at 1 MOI for indicated times (lanes 2–9). Cells were metabolically pulse-chase labeled with puromycin for 1 h prior to harvest. Cell lysates were analyzed by Western blotting with the indicated antibodies. De novo protein synthesis was assessed using anti-puromycin antibody 12D10. b HeLa cells were transfected with control plasmid, Flag-tagged NOP53, or Flag-tagged N4. The cells were infected with HSV-1/F at 0.1 MOI, and viral yields were determined 36 or 48 h.p.i. by plaque assay. These experiments were performed two times with three replicates in each experiment. Values represent means with SD. **p ≤ 0.01; ***p ≤ 0.001. c HeLa cells seeded in 24-well plate were transfected with control plasmid, Flag-tagged NOP53 or Flag-tagged N4 (500 ng each), and then infected with HSV-1/F at 0.1 MOI for the indicated times. Total RNAs were isolated; expression levels of HSV-ICP8 mRNA were quantified by qRT-PCR and normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). These experiments were performed two times with three replicates in each experiment. Values represent means with SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. d HeLa cells were transfected with siNOP or siNeg followed by infection with HSV-1/F at 0.1 MOI, and viral yields were determined 36 or 48 h.p.i. by plaque assay. These experiments were performed two times with three replicates in each experiment. Values represent means with SD. ***p ≤ 0.001. e HeLa cells seeded in 24-well plate were transfected with siNOP or siNeg (20 pmol each), and then infected with HSV-1/F at 0.1 MOI for the indicated times, and assessed as described in Fig. 3c. Values represent means of triplicates with SD. **p ≤ 0.01; ***p ≤ 0.001. f HeLa cells transfected with control plasmid (lanes 2, 4, 6, 8) or Flag-tagged N4 (lanes 3, 5, 7, 9) were mock-infected (lane 1) or infected with HSV-1/F at 10 MOI for indicated times (lanes 2–9). Cell lysates were then analyzed by Western blotting using antibodies directed against phospho-eIF2α (p-eIF2α), eIF2α, phospho-eIF4E (p-eIF4E), eIF4E, HSV-ICP8, Flag, and actin