I am analyzing the expression of a receptor tyrosine kinases protein and its phosphorylated form via Western blots. The phosphorylated form has decreased significantly, and the total remains constant after treatment. How can we explain this?
Generally, if a protein is not phosphorylated it means it is not activated by a certain protein kinase(s).
If the expression of your phosphorylated tyrosine kinase receptor (pTRK) was decreased it could be due to the inhibitory effects of protein tyrosine phosphatases.
Here is a good publication describing the effects of PTPs (DOI: 10.1038/nrm2039).
One way you can confirm that is if you treat your samples with phosphatase inhibitors. And run WB to detect treated vs. untreated phosphorylated RTKs.
See if that improves your results. Good luck.
Hi Selim
in order to explain your result : This means that your protein pTRK is down regulated >
hope this helps you
Shian
Hi Shilan,
I have not posted the question. Main author's question is above my answer.
Selim
Thank you for the responses. I used a PI3K inhibitor to block the PI3K/Akt signaling pathway in cancer cells. It is expected that an increase or decrease in the phosphorylation of the phosphorylated form would affect the total protein. As the phosphorylated form is a subset of the whole protein, how can we justify that no change has occurred in the total?
Hi Shirin,
The expression of your RTK receptor upon the treatment was not changed, however, the activity of the RTK receptor was decreased (that's why your pRTK was decreased on WB).
The decreased phosphorylation means decreased activity of the receptor. I would run ELISA activity assay kit(s).
Selim
What is the time frame of your experiment? It is possible that you could detect decreased phosphorylation of your target protein but there hasn’t been enough time to see down regulation of the total protein, especially if its a stable protein.
Hi dear Susan,
Thank you for getting back to me.
The duration of the treatment is 24 hours. The proteins under investigation are HER2 and HER3 and their phosphorylated form.
That certainly seems long enough to detect decreased total protein either by degradation or transcriptional down regulation. Could your inhibitors be blocking ubiquitinization?
I appreciate your response. I do not think so. We are utilizing a PI3K inhibitor. I am currently in the process of investigating to understand the cause of these variations. However, I have been unable to discover any relevant information thus far.
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