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. 2018 Jul 17;9:2789. doi: 10.1038/s41467-018-05176-7

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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. 7 — NDR1 binds to miR146a intergenic region to dampen NF-κB-mediated miR146a transcription. a Absolute quantification PCR analysis of pri-miR146a expression in WT and NDR1^−/− immortalized BMDMs. b Real-time PCR analysis of pri-, pre-, and mature-miR146a expression in WT and NDR1-deficient PMs. TaqMan microRNA assay using specific hydrolysis probes for mature miR146a and U6 was performed to achieve specificity. c ChIP-qPCR analysis of NF-κB enrichment in miR146a regulatory sequences with anti-NF-κB antibody in lysates of WT or NDR1^−/− PMs. d ChIP-qPCR analysis of NF-κB enrichment in miR146a regulatory sequences with anti-NF-κB antibody in lysates of NDR1 DNA binding sites deficient (NDR1 DBS^−/−) and control (NDR1 DBS^+/+) L929 cells transfected with flag-NDR1 or empty vector. e Real-time PCR analysis of mature miR146a expression in NDR1 DBS^+/+ and NDR1 DBS^−/− L929 cells transfected with flag-NDR1 or empty vector. f Immunoblot analysis of STAT1 expression in NDR1 DBS^+/+ and NDR1 DBS^−/− L929 cells transfected with flag-NDR1 or empty vector. g Immunoblot analysis of STAT1 expression in WT and NDR1^−/− PMs, transfected with miR146a inhibitor or negative control. h Immunoblot analysis of STAT1 in lysates of L929 cells stably overexpressing pre-miR146a or empty vector, transfected with plasmids expressing flag-NDR1 or flag-none. i Real-time PCR analysis of VSV-G mRNA in L929 cells stably overexpressing pre-miR146a or empty vector, treated as in h and followed by VSV infection for 8 h. j Immunoblot analysis of STAT1 in lysates of WT and miR146a^−/− L929 cells treated with NDR1-specific or scrambled siRNA for 48 h. k FACS analysis of virus replication in WT and miR146a^−/− L929 cells treated as in j, followed by VSV-eGFP infection. l Immunoblot analysis of STAT1/IRAK1/TRAF6 expression in the lysates of WT and NDR1^−/− PMs. m Immunoblot analysis of STAT1/IRAK1/TRAF6 expression in the lysate of HEK293 cells transfected with empty vector or increasing concentrations of miR146a constructs (left). Proteins densitometry was presented relative to β-actin and normalized with their densitometry on the resting state (right). Data are mean ± SD and are representative of three independent experiments. Student’s t test was used for statistical calculation. *p < 0.05, **p < 0.01, ***p < 0.001. See also Supplementary Fig. 7