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. 2022 May 19;5:483. doi: 10.1038/s42003-022-03427-4

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© The Author(s) 2022

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. 5 — a Immunofluorescence of Flag-importin α1 (Flag-Impα1) and Flag-importin α5 (Flag-Impα5) in HeLa cells transfected with AcGFP or AcGFP-ORF6. Anti-GFP and anti-Flag antibodies were used for detection of AcGFP (green) and Flag-importin αs (red), respectively. DAPI was used to stain the DNA (blue). Scale bars: 30 μm. b The graph represents the relative fluorescence values of Flag-importin αs in the nucleus compared to those of the whole cells in a. Signal intensities of total 50 nuclei from two independent experiments were measured. ***P < 0.001, two-tailed Student’s t-test. error bars represent SD. c The Flag-Impα1 or Flag-Impα5 recombinant proteins were incubated with GST-GFP and GST-GFP-ORF6 immobilized on GST-beads for 1 h, and then a pulled-down protein was collected (PD). The importin α proteins were detected using an anti-Flag antibody (WB). The bottom panel represents the proteins bound to the beads and stained with CBB. Either the Flag-Impα1 protein or the Flag-Impα5 protein was loaded as an input protein (1/20,000 dilution of the reaction). Values are kDa. d Immunoprecipitation (IP) of Flag-Impα1 or Flag-Impα5 from AcGFP-ORF6-transfected HEK293 cells following IFN-γ stimulation. HEK293 cells transfected with AcGFP-ORF6 and each Flag-importin α were treated with IFN-γ (+IFN) for 1 h. The cell lysates were incubated with anti-Flag antibody for IP. Each importin α was detected by the specific antibodies (WB). Anti-HA and anti-PY-STAT1 antibodies were used for detection of AcGFP-ORF6 and endogenous PY-STAT1, respectively. Normal mouse IgG was used as a negative control for IP. Light chain indicates the antibodies precipitated. Input was 1/400 dilution of cell lysates used for the reaction. Heavy chain appeared in the Flag-Impα5 IP sample. e Immunofluorescence of Flag-Impα1 in HeLa cells transfected with AcGFP or AcGFP-ORF6 following hydrogen peroxide (200 μM H₂O₂) treatment. Anti-GFP and anti-Flag antibodies were used for the detection of AcGFP (green) and Flag-Impα1 (red), respectively. DAPI was used to stain the DNA (blue). Scale bars: 30 μm. f The graph represents the relative fluorescence values of Flag-Impα1 in the nucleus compared to those of the whole cells in e. Signal intensities of total 80 nuclei from two independent experiments were measured. ***P < 0.001, n.s.: not significant, two-tailed Student’s t-test. error bars represent SD.