XB-IMG-139657
Xenbase Image ID: 139657
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Figure S4. Related to Figure 4. Physical mapping of Sox5 with Smad1 and Sox5 is required
to recruit Smad1 to the Msx1 and Id3 promoters.
(A) Co-immunoprecipitation examining interaction between Sox5 and Smad1 or Smad4. Lysates
from embryos expressing the noted epitope tagged proteins were immunoprecipitated (IP) with
anti-flag antibodies and immunoblotted (IB) using anti-myc antibodies. Direct immunoblotting
of lysate with either myc, flag, or HA antibodies served as input control (bottom panels). (B)
Schematic representations of Sox5 deletion constructs used in GST pulldown assays. (C) GST
pulldown assay using Xenopus embryo lysates that were either uninjected, injected with Sox5
wildtype mRNA or Sox5 deletion constructs shown in (B). Lysate was incubated with
glutathione beads coupled to GST or Smad1-GST. (D) Western blot showing the expression
levels of the Sox5 deletion proteins used in (C). (E) ChIP analysis examining Smad1 enrichment
on eEF1α, Id3 and Msx1 promoters. Smad1 enrichment is significantly diminished when Sox5 is
depleted. Embryo lysates were immunoprecipitated with antibodies against the epitope tag in
Smad1 (myc). Fold Enrichment is obtained by normalizing to uninjected sample. Error bars
represent S.E.M. of three independent biological experiments. Id3 p=0.0207, Msx1 p=0.0274.
(F) Western blot of embryo lysates injected for Smad1 Chromatin immunoprecipitation (Figure
4E,F). Three biological replicates were used and the expression for factors was analyzed for each
experiment. (G) Western blot of embryo lysates injected for Sox5 Chromatin
immunoprecipitation (Figure 4G,H). Three biological replicates were used and the expression for
factors was analyzed for each experiment. Image published in: Nordin K and LaBonne C (2014) Copyright © 2014. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View |