hey everyone and thanks in advance for your help! We are clueless about this:
I am looking at treatment response to fulvestrant and tamoxifen in breast cancer cells with specific mutations.
for that purpose i have transfected cells with FLAG-tagged ESR1 and treated with the medications before harvesting the cells for western blot. control cells had been treated with the resp. concentration of DMSO. as a control, I have also mock-transfected cells and treated them with meds or DMSO.
in mock-transfected cells ("empty" in the picture), the ESR1-band is almost gone under fulvestrant - treatment, as I expected bc fulvestrant degrades the protein.
in cells with the FLAG-ESR1 construct, the ESR1-band is extremely large under fulvestrant treatment, it is about 20 times bigger than the one in the DMSO control.
I have seen this repeatedly and I have checked the activity of ESR1 by downstream proteins. The activity of these constructs is gone under fulvestrant treatment.
--> How can I explain the presence of abundant, but inactive ESR1-protein?
Hi Laura: I'm not an expert on ESR1 or fulvestrant which is an antagonist of ESR1 as well as a proteasomal degradation accelerator, but try to explain your results: In your mock transfection, the endogenous trace amount of ESR1 not only was inhibited but degraded under fulvestrant treatment. In your positive transfectant, the amount of recombinant ESR1 was so huge (> 20 x from your estimate or even >100 in my naked eyes) that the proteosomal activities cannot be efficiently degrade them all. On the other hand, the antagonist activity of fulvestrant was so big (perhaps a very small Ki) that all of the ESR1 activities were inhibited. Solution: Try to dialyse your positive sample to see if the antagonist can be released or not prior to activity assay.
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