There seems to be some crosstalk between two signalling pathways I am looking at in breast cancer primary cells. The knockout of one receptor inhibits the activation of the other, however, the transcript levels are not altered. Does anyone know of a good approach to study this further?
Yes miRNAs could be involved, that's a good suggestion. No we aren't looking at PR/ER though. Thanks for your response!
When the transcript levels are not altered, I wouldn't think of miRNAs. It could be at the level of translation or at the level of protein stability or at the level of phosphorylation or at the level of Ubiquitination/Sumoylation and so on.
Again you are not supposed to have unaltered transcript levels when you knockout one of them. It might be either the question is not clear or the knock out is not right.
It would be better to give a bit more detail that whether the receptor mRNAs are not altered or something else to give a right suggestion..!!
Dear Sarah,
I would do a pull-down assay or co-immunoprecipitation and analyse the protein complexes with mass spectrometry. It also identifies post-translational modifications like Sumoylation, Phosphorylation, etc.!
Have you already done a simple Western blot to look at the protein expression level and/or size shifts???
Good luck,
Alex
Hi Sarah,
I agree that MS would be the best approach. If you do not have access to that you could still do an IP and check for known binding partners of either receptor. Also If they usually hetero-dimerize, that would be a straightforward explanation easily checked via IP (given you have good ABs available).
Hope that helps
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