If you want to prove the activation of the intracellular Wnt/b-catenin pathway, would an ELISA testing for the levels of intracellular phospho-b-catenin (or a ration of phospo/total-b-catenin) be enough or would you suggest additional test?
Hi,
Experiments can be included
1. TCF/LEF reporter assay
2. ELISA, Western blot, or Gene expression of Wnt ligands (WNT3a and Wnt5a), both are proven Wnt activator
3. Wnt antagonist secreted Frizzled-related proteins (sFRPs): Gene expression or Western blot
4. Wnt receptor status mainly Fzd6/7 & co-receptor LRP6
ELISA result of Phospho-beta-catenin will determine the scenario. If the Phospho-beta-catenin level decreases, then b-catenin will be active at the cytoplasm, so it can translocate into the nucleus and activate the Wnt pathway-mediated gene expression, i.e. Wnt pathway activation. And if Phospho-beta-catenin level increases then the whole process will proceed vice-versa. However, b-catenin regulation depends on death complex activity at the cytoplasm. Simultaneously, b-catenin is highly regulated by other regulators so even when the death complex is active, b-catenin may also activate & translocate to the nucleus. Therefore, if you include other components of Wnt pathways, then it will give a better answer.
Our lab also works on Wnt pathways focussing sFRPs.
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Regards
Saurabh
The below link can help to in some extent
Article Supplemental Data Wnt/??-Catenin Signaling: Components, Mech...
Top-Flash assay is one that has been most used to confirm canonical Wnt activation. You could also look at the Wnt targeting genes including Axin2, Lef1, c-myc by qPCR and/or WB.
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