We have been trying to stimulate ERK1/2 activation in MCF-7 cells (as detected by ERK1/2 phosphorylaton) with this pharmacological GPER (GPR30 ligand). So far without any success, although positive control (EGF) works pretty good. Can anybody recommend optimum exposure time or working concentration for this lignand? Or, should we select a bettter, alternative endpoint to measure? We also tried alternative activators reported in literature, such as genistein, also without any success.
- Did you check the membrane expression of GPER in your cells ? By FACS ?
You might have expression in the cytosol but the membrane expression is too low.
- I would recommend from 5 minutes to 1/2 hour. You might have very transient ERK activation. I observed that ERKs activation can be gone after 15 minutes stimulation by the ligand.
- If it is a GPCR, it can also go to the PI3K/AKT pathway. You could therefore check pAKT S473 or pAKT T308. Again I would start at short time point.
Hi Jan,
Which ligand did you use? We use G1 which is a very stong ligand (in Hela cells yet did not look at ERK activation). You might consider to strip the medium of residual estrogen (charcoal stripped medium/serum)
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