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Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
sc-12-1173_sm_SupplFigure1.pdf60KSupplemental Fig. S1. The Tcf/Lef-Oct4 composite site is essential for the activation of Mesp1 promoter by β-Catenin. Point mutations as indicted by red characters were introduced into the Mesp1-Luc reporter. CA-β-Cat failed to transactivate these mutant reporters.
sc-12-1173_sm_SupplFigure2.pdf184KSupplemental Fig. S2. The Tcf/Lef-Oct4 composite site mediates the binding of Oct4 or Lef1 to DNA. (A) The Tcf/Lef-Oct4 composite site mediates the binding between Lef1 and DNA. Lane 1: ?-32P labeled oligo probe only. Lane 2: Labeled oligo coincubated with reticulocyte lysate. The resulting band represents a non-specific banding between the labeled oligo and unknown protein in the reticulocyte lysate. Lane 3: Labeled oligo coincubated with in vitro translated Lef1. Lane 4-7: Labeled oligo coincubated with Lef1, in the presence of unlabeled “cold” wildtype or mutant oligos. While wildtype oligo and mutant oligo affecting the “octamer” side of the composite site (MtO) compete with the labeled oligo for Lef1 binding, mutant oligo affecting the Tcf side of the composite site (MtT and MtTO) showed little competition. Lane 8-9: Labeled oligo coincubated with Lef1, in the presence of Lef1, or SRF antibody. Lef1 antibody abolished the binding between the oligo and Lef1, whereas SRF antibody had no effect on the binding. (B) The Tcf/Lef-Oct4 composite site mediates the binding between Oct4 and DNA. Lane 1: ?-32P labeled oligo probe only. Lane 2: Labeled oligo coincubated with in vitro translated Oct4. Lane 3-6: Labeled oligo coincubated with Oct4, in the presence of increasing amounts of Oct4, or SRF antibody. Oct4 antibody interfered with the binding between the oligo and Oct4, whereas SRF antibody had no effect on the binding. Lane 7-10: Labeled oligo coincubated with Oct4, in the presence of unlabeled “cold” wildtype or mutant oligos. While wildtype oligo and mutant oligo affecting the Tcf side (MtT) of the composite site competed with the labeled oligo for Oct4 binding, mutant oligo affecting the “octamer” side (MtO and MtTO) of the composite site failed to compete.
sc-12-1173_sm_SupplFigure3.pdf102KSupplemental Fig. S3. β-Catenin is essential for Mesp1 expression and development of the cardiac program. β-Cat−/− ES cells were cultured as EBs to induce differentiation. RNA samples from the indicated samples were analyzed by realtime RT-PCR. N . 3; *, p < 0.05 versus control cells.
sc-12-1173_sm_Supplinfor.pdf294KSupplementary Data
sc-12-1173_sm_Suppltable.pdf7KSupplementary Table

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