Hello, I am new to Axl Receptor Tyrosine Kinase (RTK) signalling and right now I am testing one Axl inhibitor. I treated cells with this inhibitor. I observed that the downstream player of Axl signalling, p-Akt, is also inhibited, which is what I expected. However, p-Akt level starts to increase after 2h treatment. I found this hard to explain as the Axl inhibitor is always in the media. Therefore I expected the whole Axl signalling should be inhibited all the time. Can anyone help me understand why p-Akt level starts to come back after 2h inhibition of Axl?
Hi Wenchen - do you know the stability of the inhibitor at 37oC (the temperature I am guessing you are incubating your cells)?
One possibility is that the conditions (temperature) are degrading the inhibitor.
Another possibility is that the inhibitor is actively being degraded by the cells themselves (i.e. it is taken up by the cells which are then breaking it down).
A third possibility is that the inhibition is being overcome by the cells up-regulating the kinase that is phosphorylating the Axl receptor (in response to the inhibitor).
It may also be a combination of all of the above.
If you have not already done so I would suggest titrating the inhibitor upwards from the current level you are using (i.e. if using 10 ug/ml test 10, 15, 20, 50 ug/m etcl) and monitor the level of pAxl. Choose the lowest concentration that maintains inhibition for the duration of your experiment. Be aware that many kinase inhibitors are not specific and can cross react with other proteins/kinases with unexpected results (i.e. cell death).
Hope this helps,
Gary
May be using our kinome activity platform would help to get complete picture and finding any feedback loop? Contact me for more info
PamGene’s global kinase activity arrays are more sensitive (1-5 ug proteins only, 10-50k cells), robust, reproducible (
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