Fig. 7. Smad3 induces expression of Xlim1 in a direct manner; induction of Chordin by Xlim1-3m requires Smicl. (A) Smad3 but not Smad2 is a direct inducer of Xlim1. The experimental regime was identical to that described in Fig. 6A, except that expression of Xlim1 and not Chordin was analysed by quantitative RT-PCR. (B) Xlim1/3m is a direct inducer of Chordin. RNA encoding Xlim1/3m (400 pg) was injected into embryos of Xenopus laevis at the one-cell stage. Animal caps were dissected from such embryos and cultured in control medium or medium containing cycloheximide, as described in Fig. 6A. Induction of Chordin is not inhibited by cycloheximide. (C) Smicl is required for Xlim1/3-mediated induction of Chordin. RNA encoding Xlim1/3m (400 pg) was injected into embryos at the one-cell stage in the presence of 80 ng morpholino oligonucleotide coMO or XlMO. Animal pole regions were dissected from such embryos at mid blastula stage 8 and cultured until control embryos reached stage 11. (D) Smicl is not required for expression of Xlim1 in Xenopus laevis. Morpholino oligonucleotides XlMO or coMO (80 ng) were injected into Xenopus laevis embryos at the one-cell stage and RNA was extracted at early gastrula stage 10.5. Expression of Xlim1 is not inhibited by XlMO. (E) Smicl and Xlim1/3 can interact. The indicated combinations of expression constructs encoding HA tagged XtSmicl, Flag tagged Xlim1/3m, Myc tagged Smad3 and caALK4 were co-transfected into HEK 293T cells. Extracts were subjected to immunoprecipitation using an anti-Flag antibody and the presence of HA XtSmicl was analysed by western blotting by using an anti-HA antibody. Smad3 can bind Xlim1/3 in a Smic1-dependent fashion as shown by western analysis of Xlim1/3 immunoprecipitates using an anti-Myc antibody. (F) Binding of Flag-tagged Xlim and HA-tagged Smicl to Chordin promoter sequences containing Xlim-binding sites. Extracts of HEK293T cells transfected with Xlim and/or Smicl expression constructs were incubated with streptavidin-agarose beads and biotinylated double-stranded oligonucleotides. Upper panel: precipitated complexes were subjected to western blotting using anti-Flag and anti-HA antibodies. Xlim1, but not Smicl, interacts with this region of the Chordin promoter, but Smicl can form part of a ternary complex with the Chordin promoter region and Xlim1. Xlim does not bind to Chordin promoter oligonucleotides with mutations in the Xlim-binding sites (see Materials and methods). Lower panel: western blotting of whole extracts shows similar expression levels of Flag-tagged Xlim and HA-tagged Smicl.
Image published in: Collart C et al. (2005)
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