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. 2021 Apr 30;12:2482. doi: 10.1038/s41467-021-22750-8

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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. 7 — a Quantification of CFLAR protein levels in CUX1^−/− U937 cells compared with wild-type. β-Actin was used as a loading control. The experiment was performed three times. b Expression of CYBB and CFLAR transcripts normalized to HPRT1 in CUX1^−/− U937 cells compared with wild-type by qRT-PCR. Biological and technical triplicates were analyzed. c Immunoblot showing expression of biotinylated CUX1^p100 and CUX1^p200 proteins (top) in doxycycline-treated CUX1^−/− U937 cells harboring the indicated constructs. The corresponding levels of CFLAR are also shown (bottom). β-Actin was used as a loading control. The experiment was performed three times. d Promoter-luciferase assays showing luciferase activity in HEK293T cells transfected with the indicated constructs and a ~4 kb CFLAR promoter-luciferase reporter. Values were normalized to empty vector. Three experiments were performed with triplicate samples in each experiment. e Promoter-luciferase assays with deletion constructs of the CFLAR promoter-luciferase reporter (large green arrow). Blue ovals, potential CUX1-binding sites. White oval, mutated potential CUX1-binding site. TSS, transcriptional start site (nominated + 1 position). Three experiments were performed with triplicate samples in each experiment. f Sequence of potential CUX1-binding sites in CFLAR promoter closest to TSS. Locations of binding sites relative to TSS are shown. Consensus CUX1-binding site is shown (top). Red, exact consensus match. g Representative electrophoretic mobility shift assay with indicated components and proximal (−132 to −122) or distal (−156 to −146) radiolabeled, potential CUX1-binding dsDNA probes. The experiment was performed twice with similar results. h Chromatin immunoprecipitation assay followed by qRT-PCR for the CFLAR promoter region (purple) or distal control region (gray) in CUX1^−/− U937 cells complemented with the indicated constructs as shown in c. Biological and technical duplicates were analyzed. All plots show mean + s.e.m. Two-tailed, unpaired t-test.