In my project, we find one agent which can down regulate phosphorylation of 4EBP1, so we think this agent maybe can inhibit mTOR. We tried to active mTOR to check this function of agent. We used MHY1485 but the effect is very low. PA is not be applied in our lab for we do not have Lyophilizer machine. So can someone tell me how to active mTORC1 or mTOR? Or can I use some other methods to check this?
100 ng/ml IGF-1 after 16 h serum starvation has proven to amp up the mTOR pathway. I might be wrong with the concentration though.
Hormones, cytokines, growth factors and amino acids all activate mTOR. It may also be worth looking at phosphorylation of mTOR and p70S6K in addition to 4EBP1.
Good luck.
serum deprive cells in media supplemented with 1% BSA for 18-24 hrs, then treat with 10% FBS for 1, 5, 10, 20, 30 60 minutes, Stimulation with 10ng/ml PDGF for 1, 5, 10, 20, 30, 60 minutes should also stimulate mTOR
Hi!!!. You can use in deprived cells insulin 10 nM. If you want to test the inhibitory Effect of your molecule, you could use as positive control rapamycin 40nM.
Good luck!!!!!
Try C8 PA from Avanti. It's very water soluble so it should go into solution with vortexing. Even better would be to bath sonicate the solution for 3 min.
Here is an example of how we would prep a stock solution for stimulating cells in a 6 well dish.
** Stimulate 4 wells with 30 µM C8 PA using 50µL of solution per well. The media volume is 1mL / well, so add 0.03 µmol of PA (30 µmol/L = 0.030 µmol/mL) per well. To do this make 225 µL of solution with 0.135 µmol of PA in 225 µL of PBS.
Stock C8 PA in chloroform... C8 PA MW = 446.45g/mol =446.45µg/µmol *0.135 µmol = 60.27µg = 0.06027 mg (stock = 10mg/mL, so need 6.03µL of stock PA ). Take the 6.03µL of PA and dry with stream on nitrogen, add PBS and bath sonicate for 3 min. Make it immediately before use.
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