XB-IMG-42245
Xenbase Image ID: 42245
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Fig. 2. Localisation and activity of XtSulf2. RNAs coding for tagged proteins were injected into the animal hemisphere of both blastomeres of a two-cell X. laevis embryo and processed for immunohistochemistry at NF stage 10. (A) Embryos injected with 1 ng of Nuc-GFP. (B and C) Embryos injected with 2 ng of XtSulf2–GFP. (D–F) Embryos co-injected with RNA coding for XtSulf1-Myc (D) and XtSulf2–GFP. (E) The yellow regions in (F) show where XtSulf1 and XtSulf2 expression overlap. Embryos were co-injected with mRNA coding for FGFR1-Myc (G) and XtSulf2–GFP (H) proteins. While both proteins appear associated with the membrane, they do not completely overlap (I). Western analysis shows in (J) that XtSulf2 while injection of mRNA coding for XtSulf2 inhibits activation of dpERK by FGF4 protein, it has no effect on dpERK activation by the intracellularly drug induced iFGFR1. (K) BMP4 activity was assayed by Western blot analysis using antibodies against phospho-SMAD 1/5/8. The phosphorylation of SMAD 1/5/8 stimulated in animal caps by injection of mRNA coding for BMP4 is down-regulated in animal caps co-expressing XtSulf2. However, animal caps expressing a constitutively active BMP4 receptor, Alk3 continue to express phospho-SMAD 1/5/8 in the presence of XtSulf2, indicating that XtSulf2 acts upstream of the BMP4 receptor. (L) Neuralisation of animal caps was assayed using RT-PCR to detect NCAM expression. Whole embryos (WE) express NCAM at NF stage 18, while control animal caps do not. Injection of mRNA coding for XtSulf2 results in the activation of NCAM expression. Samples with water and without AMV Reverse Transcriptase (RT) were used as negative controls; L8 was used as a loading control. Image published in: Guiral EC et al. (2010) Copyright © 2010. Image reproduced with permission of the Publisher, Elsevier B. V.
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