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Research Article
Differential requirements for Smad4 in TGFβ-dependent patterning of the early mouse embryo
Gerald C. Chu, N. Ray Dunn, Dorian C. Anderson, Leif Oxburgh, Elizabeth J. Robertson
Development 2004 131: 3501-3512; doi: 10.1242/dev.01248
Gerald C. Chu
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N. Ray Dunn
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Dorian C. Anderson
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Leif Oxburgh
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Elizabeth J. Robertson
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Data supplements

  • DEV01248 Supplemental Figure

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      Fig. S1. Functional characterization of the Smad4Nallele. Smad4N/N ES cells were generated by targeting the remaining wild-type allele in Smad4N/+ES cells with the Smad4TA replacement vector followed by Cre-mediated excision of exon 1 (Fig. 1A). (A) Western blot analysis of ES cell and HepG2 hepatoma cell protein extracts with a Smad4 monoclonal antibody. A 64 kDa immunoreactive band is seen in wild-type CCE ES cells, Smad4N/+ ES cells and control HepG2 cells but not Smad4N/N ES cells. (B) Semi-quantitative RT-PCR analysis of Smad4 target genes in embryoid bodies (EBs). RNA was isolated from wild-type (+/+), heterozygous (N/+) and homozygous (N/N) ES cells on days 5, 7, 10 and 12 of differentiation. Genes examined were Msx1 and Msx2, Smad4-dependent targets of Bmp signaling, and the visceral endoderm markers, Hnf4 and transferrin. Gapd was used as a normalization indicator. The marked downregulation of these genes in Smad4N/N EBs was identical to that described for the previously characterized Smad4tm1Ari null allele (Sirard et al., 1998; Sirard et al., 2000). (C) Smad4N/N murine embryonic fibroblasts (MEFs) were generated for reporter assay analysis using a tamoxifen-inducible Cre transgene (Hayashi and McMahon, 2002). Wild-type (black bars) and Smad4-deficient (grey bars) fibroblasts were transfected in triplicate with 3TP-lux and stimulated with Tgfb1 ligand. A Tgfb response was detected in wild-type, but not Smad4N/N MEFs. Separately, constitutively active Bmp type I receptor (caALK3) was transfected together with Msx2-lux. A Bmp-response was detected only in wild-type cells. Ligand-responsiveness was restored to Smad4N/N MEFs by co-transfection with a Smad4 expression construct (pCMV-MADR4, white bars). These Smad4-dependent Tgfb and Bmp responses reproduce those observed in Smad4tm1Ari null MEFs (Sirard et al., 2000).

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Research Article
Differential requirements for Smad4 in TGFβ-dependent patterning of the early mouse embryo
Gerald C. Chu, N. Ray Dunn, Dorian C. Anderson, Leif Oxburgh, Elizabeth J. Robertson
Development 2004 131: 3501-3512; doi: 10.1242/dev.01248
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Research Article
Differential requirements for Smad4 in TGFβ-dependent patterning of the early mouse embryo
Gerald C. Chu, N. Ray Dunn, Dorian C. Anderson, Leif Oxburgh, Elizabeth J. Robertson
Development 2004 131: 3501-3512; doi: 10.1242/dev.01248

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