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Expression of DUX4-fl in human tissues.

By Lauren Snider (360240), Linda N. Geng (360242), Richard J. L. F. Lemmers (360244), Michael Kyba (121283), Carol B. Ware (360247), Angelique M. Nelson (360249), Rabi Tawil (169550), Galina N. Filippova (169552), Silvère M. van der Maarel (1389870), Stephen J. Tapscott (269326) and Daniel G. Miller (169553)

Abstract

<p>(A) RT-PCR of RNA from human tissues showing DUX4-fl in the testis sample (Testis-1) and DUX4-s in ovary heart and liver. Note that each sample is from an unknown individual and their genotype is not known. (B) RT-PCR of three additional testis samples and matched skeletal muscle RNA from the Testis-1 and Testis-4 donors. (C) RT-PCR of the full-length DUX4 ORF (nested with 36 total cycles) in muscle (M) and testes (T) RNA from the same individuals, showing expression in testes and not in muscle. Numbers indicate Testis-1, 4, 7, 6, 5. (D) Quantitative RT-PCR of DUX4-fl3′ showing relative abundance in muscle cells, muscle biopsies, human testis and othr indicated cells. The sample with the highest expression was set at 100% and the number of copies per cell roughly estimated based on titration of input DNA. H9-ES, human embryonic cell line H9; M83-9 iPS, iPS line made from control fibroblasts; 43-1 series are the fibroblast, iPS, and EB cells from an FSHD fibroblast line; MB183 and MB148, FSHD muscle cultures under growth (MB) and differentiation (MT) conditions; F-2331 and F-2316, FSHD muscle biopsy samples; hTestis, human testis. (E) Western detection of DUX4 protein in whole cell extracts from tissues and cell lines using a rabbit monoclonal antibody (E14-3) raised to the aminoterminus of the human DUX4 protein. Lanes: 1-control muscle culture; 2-HCT116 cell line; 3-mouse testis; 4-human testis; 5-C2C12 cells transfected with human DUX4 expression vector; 6-C2C12 cells. Specificity of the antibody is indicated by selective detection of DUX4 protein in C2C12 cells transfected with a DUX4 expression vector, pCS2-DUX4 (note that this vector contains two ATG codons resulting in the standard DUX4 protein and an in-frame slightly larger protein accounting for the two bands on the western), compared to untransfected C2C12. Human testis has a single reactive band that migrates marginally slower than the transfected standard DUX4 species in C2C12 cells. This DUX4-reactive band is not present in mouse testis extract (note that mice do not have a highly conserved DUX4), HCT116 human colon cancer cells, nor unaffected myotubes (MB196-36hr). Similar results were obtained with an additional DUX4 antibody (E5-5) raised to the carboxyterminal region of DUX4 (data not shown)) and on protein extracts from two additional testis samples (data not shown). (F) Immunoprecipitation of indicated protein extracts with the E14-3 rabbit monoclonal to the N-terminal region of DUX4 followed by western with the P4H2 mouse monoclonal to the C-terminal region of DUX4 demonstrating that the protein recognized by the rabbit anti-DUX4 is also recognized by an independent mouse monoclonal to DUX4. Lanes: 1-HCT116 cell line lysate; 2- Testis protein lysate; 3-C2C12 cells transfected with DUX4 expression vector.</p

Topics: Cell Biology, Genetics, Neuroscience, Developmental Biology, dux4-fl
Year: 2013
DOI identifier: 10.1371/journal.pgen.1001181.g003
OAI identifier: oai:figshare.com:article/491515
Provided by: FigShare
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