Effects on basal S6 phosphorylation could be seen with rapamycin concentrations as low as 25-100nM (We use 50nM usually and monitor s6 phosphorylation as control).

Regarding the second part, the best option for mTORC2 inhibition would be a siRNA based approach. If thats not possible, now there are some mTORC1/2 inhibitors whose effects could be compared with that of low concn rapamycin to get some information regarding differential role of the complexes. Good Luck,

Missed the time factor in your question- effect on basal S6 phosphorylation we could see at 2h treatment and was effective when we checked at 24h also. p70-S6K phosphorylation could be a better marker than phospho-S6. Hope it helps

I would advise you to use a dose curve of rapamycin, for example 0.1, 0.3, and 1 µM.

You may find useful our work we published:

A. Moumen, S. Patané, A. Porras, R. Dono and F. Maina. mTOR acts through Mdm2 to signal cell survival in vivo. Development 134:1443-51 (2007).

Good luck!

Flavio

I see suppression of S6K1 Thr389 phosphorylation and phospho-rpS6 after 1 hr treatment at 50nM rapamycin in multiple cell types. mTORC1 has rapamycin insensitive effects, rapamycin can also repress mTORC2 complex formation after long term treatment and mTOR kinase inhibtors repress both complexes so the only real way to distinguish is knockdown of raptor/rictor..

Thanks guys your suggestions were really useful. I used 100nM Rapamycin for 4 hrs and saw a decrease in p-s6k. I am also trying to use AZD8055 for mTORC2 inhibition anyone in this forum has any experience working with this drug.

Maybe that our paper: "Inhibition of mTORC1 by SU6656, the selective Src kinase Inhibitor, is not accompanied by activation of Akt/PKB signalling in melanoma cells", by L. Ondrušová, J. Réda , P. Žáková, Z. Tuháčková, published in Folia Biologica 59, 162-167 (2013),.could be useful for the solution of your problem. Good luck!