XB-IMG-23557
Xenbase Image ID: 23557
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Figure 1. Smad1 Is Phosphorylated and Inhibited by GSK3(A) Smad1 contains MAPK (blue) and GSK3 (red) phosphorylation sites in its linker region. The PPAY binding site of Smurf1 is boxed and serines 210 and 214 used to raise antibodies indicated by asterisks.(B) Smad1 constructs encoding Smad1 wild-type (SWT) or phosphorylation-resistant mutants for GSK3 (SGM) and MAPK (SMM) sites.(C–F) Injection of SGM or SMM, but not of SWT, mRNA increased expression of the ventral marker sizzled in Xenopus embryos.(G) A BMP-independent phospho-mimetic activated Smad1 (SEVE) in which the SVS terminus was mutated into EVE.(H–J) Activity of SEVE is increased by phosphorylation-resistant linker mutations.(K) GSK3 radioactively phosphorylates Smad1 in vitro, but only when primed by MAPK. The recombinant Smad1 linker substrate was about 90% pure in polyacrylamide gels (data not shown).(L and M) Phospho-specific antibodies for hSmad1 Ser214 (pSmad1MAPK) and Ser210 (pSmad1GSK3 antibody B was used).(N) Phospho-specific antibodies (pSmad1MAPK and pSmad1GSK3-A) demonstrate that GSK3 phosphorylation of recombinant Smad1 requires MAPK priming, in nonradioactive Western blots. Recombinant Smad1 substrate was used in the same amount as in (K). Image published in: Fuentealba LC et al. (2007) Copyright © 2007. Image reproduced with permission of the Publisher, Elsevier B. V.
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