Figure 5
Acute glutamine depletion and ammonia lead to dynamic changes in mTORC1 and AMPK signalling in HEK293 and Huh7 cells. HEK293 and Huh7 cells were plated in complete media containing glutamine for 24 h. One culture was collected for western blotting 24 h later (T0). The remainder of the cultures were washed with DPBS and fresh glutamine-depleted media with or without 0.8 mM NH4Cl was added. Cultures were collected at different time points and protein was extracted for western blotting. Protein abundance of phosphorylated S6K (T389) relative to total S6K protein levels (A,C) and phosphorylated ACC (S79) relative to total ACC protein levels (B,D) at 15 min, 30 min, 1 h, 2 h, 4 h and 6 h post-incubation was quantified using western blot. Blots shown were representative from 3 independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Full length blots are presented in Supplementary information Fig. 4.
