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. 2021 Nov 15;12:6602. doi: 10.1038/s41467-021-26910-8

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© The Author(s) 2021

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 Effect of SARS-CoV-2 proteins on NLRC5-mediated HLA-B promoter activity. The schematic image illustrates the reporter assay system for monitoring the promoter activity of HLA-B. HEK293T cells were transfected with HLA-B proximal promoter reporter construct and plasmids expressing WT or Walker A mutant NLRC5, along with a control plasmid, or with plasmids expressing the indicated SARS-CoV-2 proteins. At 36 h after transfection, cells were collected to measure the luciferase activity. The results are from three independent experiments. b Effect of SARS-CoV-2 ORF6 on NLRC5-mediated expression of the MHC class I-related genes in HEK293T cells analyzed by quantitative real-time PCR. The results are from three independent experiments. c Effect of SARS-CoV-2 proteins on NLRC5-mediated surface expression of HLA- A, B, and C in HEK293T cells analyzed by flow cytometry. GFP (NLRC5) positive cells were gated for evaluating PE (HLAs) signal intensity. The FACS gating strategies are provided in Supplementary Fig. 9. d Effect of SARS-CoV-2 proteins on the cellular localization of FLAG-tagged NLRC5 in leptomycin B (100 nM, 8 h) treated HeLa cells stably expressing FLAG-NLRC5 by immunofluorescence analysis. Representative images show reduced NLRC5 nuclear localization indicated with white arrows. Quantitative comparison of the nuclear NLRC5 signal intensity (% of nuclear signal intensity/total cell signal intensity) is shown with a bar graph analyzed by ImageJ. Images were obtained using a confocal microscope. The scale bar indicates 50 microns. The sample numbers for evaluation are indicated in the bar graph. e Immunoblot analysis of the indicated proteins from the cell lysates of the nuclear or cytosolic fraction in either control or SARS-CoV-2 ORF6 expressing FLAG-NLRC5 HeLa stable cells. Asterisks indicate two distinct bands of NLRC5. Quantitative comparison of the cytosolic or nuclear FLAG-NLRC5 signal intensity normalized by the intensity of Lamin B1 (for nuclear NLRC5) or α/β-tubulin (for cytosolic NLRC5) is shown with a bar graph via analysis with ImageJ. The data shown is a representative result from three independent experiments.