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. 2018 Jul 23;4:70. doi: 10.1038/s41420-018-0072-3

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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, visithttp://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Sanger sequencing and sequencing alignment of FH (E404D) (NCBI reference sequences are shown on the right). Black arrow indicated that the heterozygous mutation in FH (c.1212G>C, p.Glu404Asp) was further confirmed by Sanger sequencing. C.elegans Caenorhabditis elegans, D.melanogaster Drosophila melanogaster, E.coli Escherichia coli, S.cerevisiae Saccharomyces cerevisiae, S.tuberosum Solanum tuberosum. b FH Glu404Asp significantly compromised enzyme activity by overexpressing recombinant FH-Myc protein in HEK 293T cells in vitro (***P < 0.001). An empty vector served as a control (Ctrl). Relative activity is presented as the mean ± SE of four independent experiments; each sample was assayed in quadruplicate in each experiment. c Expression of FH was downregulated, whereas G6PD (glucose-6-phosphate dehydrogenase) were upregulated in the cardiac tumor tissue when compared with adjacent normal tissue. GAPDH and β-ACTIN served as loading controls. Nt adjacent normal tissue, Tt tumor tissue. d FH E404D significantly increased levels of cellular fumarates in vitro (**P < 0.01). HEK 293T cells were transfected and an empty vector served as a control. e FH (E40ED) affected normal tetramer formation. HEK 293T cells were transfected with Myc-tagged wild type or (E40D) FH for recombinant overexpression. A 1:100 dilution of a protein crosslinker (PC) was added to the RIPA buffer before cell lysis. Subsequently, anti-Myc immunoprecipitation and anti-FH immunoblotting were performed following the standard procedures. A 50-KDa band was indicative of FH-Myc recombinant protein. After PC treatment, a 220-KDa band (black arrow) was generated, corresponding to an FH tetramer. This band was significantly decreased in cells transfected with mutant FH. f Native gels assay was performed to test the polymer formation of FH. After anti-Myc immunoprecipitation, two different bands were generated. The higher band, corresponding to polymer, was decreased in the cells transfected with mutant FH