Recently, we are testing mTOCR activity, by using 4EBP(Cell Signaling Technology #9452) and PP70 antibody(Cell Signaling Technology #9205), we have some problems. 1. there is always many background with PP70 antibody.
2.First we starve cell with EBSS 1h, then after EAA(complete medium) treatment, we do see the phosphation of 4EBP , but we also observe the total amount of 4EBP do down after EAA treatment, which seems abnormal and different with literature report. Do someone has experience with mTORC antibody, or those two antibodies, thanks.
I have used phospho-P70S6K(T421/S424), CST, #9204 and total P70S6K (CST, #9202) before to assess mTORC1 activity which seem to work quite well. I have also tested phospho-P70S6K(T389), CST, #9205 before but found it to detect more weakly than #9204 - although it gives the same detection pattern in the presence/absence of stimulation.
It may also be beneficial to use a negative control to verify that the insulin you are using is working properly under these culture conditions (e.g. treat the cells with Rapamycin in complete media). Alternatively, you may want to use engineered, recombinant IGF (I1271, SIGMA) instead, which is more potent and stable compared to insulin.
Hi Kecheng,
I heard that there are many changes in mTOR activity that are not straight forward. I once worked on the involvement of mTOR in autophagy (please see https://www.researchgate.net/publication/260112146_MTORp70S6K_signaling_distinguishes_routine_maintenance-level_autophagy_from_autophagic_cell_death_during_influenza_A_infection OR http://www.sciencedirect.com/science/article/pii/S0042682214000117) and I was a bit confused of what the data meant too. Hopefully our work above, related or not to yours, will be of help! Best of luck!
-ED
Article MTOR/p70S6K signaling distinguishes routine, maintenance-lev...
Hi Jason Shu,
Thanks a lot for your idea. Good to know this antibody works for you. May I ask, during you treatment (presence or absence stimulation) and starvation progress, did you add some proteinase inhibitor inside? because I always observe the total amount of 4EBP decrease. Thanks.
Best,
Kecheng
Hi Emmanuel Datan,
Thanks for your nice paper, I went though it, very interesting. I need to talk with my supervisor about this. Thanks a lot.
May I ask, during you treatment (presence or absence stimulation) and starvation progress, did you add some proteinase inhibitor inside? because I always observe the total amount of 4EBP decrease. Thanks.
Best,
Kecheng
Hi Kecheng,
I feel that P70S6K is probably a better and more stable indicator of mTORC1 activity opposed to 4EBP since it is either phosphorylated or unphosphorylated with no change in total protein. Since mTORC1 activation stimulates cell growth, if nutrients are scarce (i.e. starved conditions), it is likely that the cell downregulates translation-associated proteins such as 4EBP by reducing the transcription of the gene. I wouldn't add any proteinase inhibitor though since that will just complicate the interpretation of your results (inhibitor will just cause all proteins to be stabilised).
It's probably better just to try a different P70S6K antibody - as for any phospho-protein study it is essential that you add phosphatase inhibitor to the cell lysate prior to WB otherwise you may lose the phosphorylated signal.
Article PI3K/mTORC2 regulates TGF-b/Activin signalling by modulating...
Hi Jason Shu Lim Yu,
yes, your phosphatase inhibitor is a good point, we didnot add it. Maybe this is the reason that we always the decrese of total 4EBP, maybe because we lose the phosphorylated signal.
We tried many time with P70S6K antibodies, always many noise in the background. we are still optimizing the setting of this antibody... Thanks.
Best,
Kecheng
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