Figure 4.
Effect of early mTOR inhibition on PI3K/mTOR and ERK signaling pathways. Rats were implanted with 21-day slow release rapamycin or placebo pellet on day 21 of the Solt-Farber protocol and euthanized 70 days post-DENA administration. (A) Immunohistochemistry for GST-P and phospho- S6Ser235/236 was performed on consecutive sections. Shown are representative Aperio scans acquired at 5×. Scale bar: 200 µm. (B) Total liver homogenates were prepared from rats placed on the Solt-Farber protocol, administered rapamycin or placebo via a 21-day slow release pellet and euthanasized on day 70 post-DENA administration. Homogenates were analyzed by immunoblotting with antibodies against total 4E-BP1. (C) Total liver homogenates were prepared from control adult rats (Ctl) and from rats placed on the Solt-Farber protocol and administered rapamycin via a 21-day slow release pellet. All rats were injected with either EGF plus insulin or vehicle (placebo control) 15min prior to euthanasia on day 70 post-DENA administration. Homogenates were analyzed by immunoblotting with antibodies against phospho-S6(Ser235/236), total S6, phospho-Akt(Thr308), phospho-Akt(Ser473), total Akt, phospho-Erk1/2(Thr202/Tyr204) and total Erk1/2. Densitometric analysis of the basal activity is shown in the graphs as mean + 1SD for the ratio of phospho- to total for each protein. n ≥ 4 animals per group. There were no significant differences identified using an unpaired t-test.