Figure 6.

Trib3 regulates phosphorylation of the Akt substrate FoxO1a. (a–d) Trib3 overexpression is sufficient to reduce phosphorylation of FoxO1a in neuronal cells. (a and b) Neuronal PC12 cells and (c and d) SCG neurons were infected with either control or Trib3 expressing lentivirus and maintained with NGF. Five days after infection, cell lysates were subjected to SDS-PAGE and immunoblotted using antisera against phospho-FoxO1a, total FoxO1a, Trib3 and total ERK. Panels (a) and (c) show representative blots; panels (b) and (d) show mean values for protein levels±S.E. for four independent experiments, in each case normalized to total ERK levels. Panel (d) represents mean values for protein levels normalized to total FoxO1a levels, as well. (e and f) Trib3 contributes to FoxO1a dephosphorylation after NGF deprivation. SCG neurons were infected with lentivirus expressing shTrib3 or control shRNA. Seventy-two hours post infection, NGF was removed for 16 h, and the neurons were lysed and lysates were subjected to SDS-PAGE and immunoblotted using the antisera described above. Panel (e) shows representative blots; panel (f) shows mean values for protein levels±S.E. for 8 independent experiments, in each case normalized against total ERK. *P<0.05, **P<0.005; Student's paired t-test