XB-IMG-149983
Xenbase Image ID: 149983
|
Figure S2. Noggin4 cannot antagonise BMP and Activin/Nodal signalling but inhibits Wnt/b-Catenin pathway. (a)
Comparison of Noggin1 and Noggin4 abilities to bind BMP4, using immunoprecipitation. (b) In contrast to Noggin1 and 2,
Noggin4 does not cause reduction of endogenous phosphorylation of Smad1. Embryos were injected with Noggin1/2 or
Noggin4 mRNA (100pg/embryo) and phosphoSmad1 was detected on Western-blot by anti-phosphoSmad1 antibody (Cell
Signaling Technology, Inc.). Overall level of Smad1 was detected by anti-Smad antibodies (ab66737, Abcam). Alpha-tubulin
serving as a loading control was revealed by anti-tubulin antibodies (DM1A, Sigma). In all these cases, Fab fagments of
Anti-Rabbit antibodies conjugated to alkaline-phospotase (A3937, Sigma) were used for detection of primary antibodies. (c)
Effects of Noggin1, Noggin2 and Noggin4 on expression of the BMP-specific luciferase reporter, BRE. Embryos at two-cell
stage were injected with BRE plasmid, either alone (control) or mixed with Noggin1 or Noggin2 mRNAs taken in a fixed
concentration, or Noggin4 mRNA taken in increasing concentrations. (d and e) Effects of Noggin4 on expression of the
Smad2-specific reporter ARE activated by ActivinB or Xnr2. (f) The effect of Noggin4 on expression of the
Wnt/b-Catenin-specific reporter TOPFlash activated by Wnt8 and b-Catenin . (g) qRT-PCR analysis of expression of the direct
genetic targets of the canonical Wnt pathway: Axin2, HoxA1, HoxB1, HoxD1, Siamois and Xnr3, in the late gastrula embryos
(stage 12) injected at 4-cell stage with Noggin4 mRNA or Noggin4 MO1. For each of the tested genes, the expression level in
the wild-type embryos was taken as one unit. Image published in: Eroshkin FM et al. (2016) Copyright © 2016, Macmillan Publishers Limited. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |