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. 2020 Sep 22;48(18):10479–10499. doi: 10.1093/nar/gkaa765

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© The Author(s) 2020. 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 7. — HTLV-1 IRES activity is stimulated by PRMT5 activity and PRMT5-associated hnRNP A1 methylations. (A-B) hnRNP A1-HA mutant plasmids (500 ng) for the phosphorylation sites S4/S6 and S199 (indicated in the x-axis) were co-transfected with the dl HTLV-1 IRES plasmid (300 ng). Total protein extracts were prepared 24 hrs post-transfection. (A) Western blots were performed to detect the expression level of total hnRNP A1 and hnRNP A1-HA proteins using GAPDH as a loading control. (B) RLuc and FLuc activities were measured, and results are presented as RTA, relative to the activities obtained from the dl HTLV-1 IRES vector in the presence of the wt-hnRNP A1 protein, set to 100%. Statistical analysis was performed by an ordinary one-way ANOVA test (*P <0.05). (C, D) Cells transfected with the dl HTLV-1 IRES (300 ng) plasmid were treated, or not (–), with EPZ015666 (1, 5 or 10 μM). Total protein extracts were prepared 48 hrs post-transfection and treatment. (C) The levels of PRMT5 and hnRNP A1 proteins and PRMT5 activity (SDMA) were determined by western blot using GAPDH as a loading control. (D) RLuc and FLuc activities were measured, and data are presented as RTA relative to the untreated (−) condition set to 100%. Statistical analysis was performed by an ordinary one-way ANOVA test (*P<0.05). (E, F) The dl HTLV-1 IRES (300 ng) was co-transfected with the wt-hnRNP A1-HA (500 ng) or hnRNP A1-HA R218K/R225K (500 ng) encoding plasmids. Total protein extracts were prepared 24 h post-transfection. (E) Western blots were performed to detect the levels of endogenous and overexpressed hnRNP A1-HA proteins, using GAPDH as a loading control. (F) RLuc and FLuc activities were measured, and data are presented as RTA relative to the wt-hnRNP A1-HA, set to 100%. Statistical analysis was performed by an unpaired two-tailed t-test (*P<0.05). Values shown are the mean (±SEM) for three independent experiments, each performed in duplicate. The relative level of total hnRNP A1 protein (%) in (A, E) was estimated based on the intensity of immunoreactive bands by imageJ.