Wnt5A is a non-canonical wnt ligand that activates one or more of the known non-canonical wnt signaling pathways. One of these pathways is the Wnt/PKC pathway. I need to quantify PKC activation from a Western blot, but I don't know which antibodies to use. There are 15 isozymes of PKC, each with an antibody. For those 15 isozymes there are also multiple phosphorylation states, each with its own phosphoAntibody. I can't afford 30+ antibodies, and I'm having a lot of trouble finding anything in the literature about this. Does anyone know which isozymes are associated with non-canonical wnt signaling, or which antibodies I should use?
Hi,
Colleague of mine used PKC alpha and delta (in IF, not tried in WB)...for non canonical Wnt signaling, and our Ab are from cell signalling.
bye
Hi Rahul,
Did you colleague use a specific phosphoPKC delta Antibody? I don't see one from Cell Signaling.
Many thanks!
Hello Carl,
I am sorry, there was a mistake.PKC alpha is cell signaling but for delta, we performed overexpression studies and used a tagged protein.
sorry again
bye
Another way to go is to determine the level of PKC substrates in the cell. Use the PKC substrates antibody from Cell Signaling Technologies. This antibody reports on any protein that has a phosphorylated PKC consensus site, rather than determining which isoform is activated (which could be more than one). Do the experiment with and without a PKC inhibitor
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