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FIGURE 8. XSMOC-1 inhibits BMP2 activity, but not by direct ligand binding. A, embryos were injected bilaterally at the two-cell stage with 360 pg of GFP (control), 60 pg of BMP2 plus 300 pg of GFP, or 60 pg of BMP2 plus 300 pg of XSMOC-1 mRNAs and incubated until stage 26. In three independent experiments, BMP2-injected embryos were ventralized (82% ≤ dorso-anterior index of 1, n = 84), whereas those co-injected with BMP2 and XSMOC-1 showed partial to complete rescue (70% ≥ dorso-anterior index of 3 or greater, n =98). B and C, RT-PCR analysis of animal cap explants removed from embryos at stage 8 and cultured until sibling embryos reached stage 17. B, RT-PCR for the ventral marker XVent-1 was induced in caps over-expressing BMP2 but not in control injected caps or caps co-expressing BMP2 and XSMOC-1. C, RT-PCR analysis of animal cap explants removed from embryos injected bilaterally at the two-cell stage with 400 pg of GFP (control), 100 pg of activin plus 300 pg of GFP, or 100 pg of activin plus 300 pg of XSMOC-1 mRNAs incubated until stage 17. Expression of the mesodermal marker Brachyury (Bra), induced in caps overexpressing activin, was not inhibited by co-expression of XSMOC-1. D, immunoblot analysis of mouse 3T3 fibroblast cell lysates. 3T3 fibroblasts were transfected with or without XSMOC-1 and exposed to BMP2 for 1 h. Phosphorylation of Smad1, -5, and -8 by BMP2 was blocked in cells transfected with XSMOC-1. E, RT-PCR analysis of animal cap explants removed from embryos injected bilaterally at the two-cell stage with 450 pg of GFP (control), 150 pg of caBMPR1B plus 300 pg of GFP, or 150 pg of caBMPR1B plus 300 pg of XSMOC-1 mRNAs were incubated until stage 17. The expression of the ventral marker XVent-1, induced by over-expression of constitutively active BMP receptor IB (caBMPR1B), was blocked by co-expression with XSMOC-1 but not by noggin. -RT, without reverse transcriptase. "This research was originally published THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.284(28) pp.18994-19005, July 10, 2009. Copyright: The American Society for Biochemistry and Molecular Biology." Image published in: Thomas JT et al. (2009) Copyright © 2009. Image reproduced with permission of the publisher 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

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