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. 2021 Dec 6;50(1):411–429. doi: 10.1093/nar/gkab1188

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© The Author(s) 2021. 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 (https://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 5. — Staufen1-RNA interaction is required to promote HIV-1 IRES activity. HEK 293T cells were cotransfected with the dl HIV-1 IRES (300 ng) and different quantities (50–650 ng) of a plasmid encoding for a Stau1⁵⁵⁻F135A-HA protein. (A) The overexpression of Stau1⁵⁵⁻F135A-HA protein was confirmed by western blot using an anti-HA antibody and GAPDH as a loading control. (B, C) RLuc and FLuc activities were measured 24 h post-transfection, and results are presented as RLA (B) or RTA (C). The RLA and RTA values obtained in the absence (−) of the Stau1⁵⁵⁻F135A-HA expressing plasmid were set to 100%. Values shown are the mean (±SEM) of four independent experiments, each performed in duplicate. Statistical analysis was performed by an ordinary one-way ANOVA test. (D) Schematic representation of the TAR-deletion mutants generated for the study based on mutants used to study Stau1⁵⁵-TAR interaction (33). TAR corresponds to the wild-type RNA structure. The Staufen1 binding site (SBS) is highlighted (33). The highlighted areas in TAR-mu1, TAR-mut2, and TAR-mut3 correspond to the deleted nts. Mutants were verified by sequencing. (E) HEK 293T cells were transfected with the dl HIV-1 IRES, dl HIV-1 IRES TAR-mut1, dl HIV-1 IRES TAR-mut2, or dl HIV-1 IRES TAR-mut3 (300 ng) plasmids. RLuc (upper panel) and FLuc (middle panel) activities were measured 24 h post-transfection, and data are presented as RLU or as RTA (lower panel). (F, G) HEK 293T cells were cotransfected with the dl HIV-1 IRES, dl HIV-1 IRES TAR-mut1, dl HIV-1 IRES TAR-mut2, or dl HIV-1 IRES TAR-mut3 (300 ng) and 650 ng of a plasmid encoding for a Stau1⁵⁵-HA₃ protein. (F) The overexpression of Stau1⁵⁵-HA₃ was confirmed by western blot using an anti-HA antibody and GAPDH as a loading control. (G) RLuc and FLuc activities were measured 24 h post-transfection, and results are presented as RTA were the RTA obtained with the dl HIV-1 IRES in the absence of Stau1⁵⁵-HA₃ was set to 100% (±SEM). Values shown are the mean (±SEM) of three independent experiments, each performed in duplicate. Statistical analysis was performed by an ordinary one-way ANOVA test.