Does anyone have a good protocol for human fibroblasts starvation in order to inactivate the mTOR pathway and check phospho-S6 ribosomal protein expression?
See our paper in the link below.
Link: http://www.ncbi.nlm.nih.gov/pubmed/21573174
I'm not sure what your experimental design is, but starvation will cause perturbations in PKA, Akt, AMPK, and certain PKC pathways (among others). If you want to minimize this crosstalk you can directly inhibit mTOR activity using rapamycin (sirolimus).
Alternatively, if you'd like the full physiological effects of starvation, there are multiple protocols for serum starvation of fibroblasts (links below).
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4082808/
http://www.ncbi.nlm.nih.gov/pubmed/19788523
I try to induce the activation of mTOR pathway in human fibroblasts by amino acids starvation.
I try differents conditions : 1 to 3 hours in EBSS and cultured medium during 10 min to 2 hours.
I'm look by IHC if mTOR colocalyse with LAMP2.
I don't have any results.
Did someone have the good times of starvation to see mTOR activation ?
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