I cloned a Xenopus Notum homolog. Its sequence is identical to the published xNotum. It is tagged with a 3X Flag. When I co-inject it together with Wnt8 into ventral blastomeres of Xenopus embryos, it does not inhibit Wnt-induced double axis,meaning it does not antagonize Wnt. Did Anyone have a similar experience?
You could assay a prepared cell lystate for depalmitoleyation of wnt to see if it is exerting its biological effect and that notum is not being inappropriately trafficked or targeted in the cell (possible a consequence of the HA tag). You could technically look where the protein is targeted by immunofluorescence.
Dear Carolyn, indeed I thought that the 3Xflag could affect the Notum activity, so I changed it with a myc tag and it seems to work better, although I need to characterize it better. I'll probably turn to cell culture for some tests. Thank you for your answer!! :)
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