Figure 5.

Trib3 regulates neuronal Akt phosphorylation. (a–d) Trib3 overexpression is sufficient to reduce Akt phosphorylation in (a and b) neuronal PC12 cells and (c and d) SCG neurons. Cells 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-Akt (pSer473 and pThr308), Trib3, total Akt and total ERK. Representative immunoblots are shown in panels (a) and (c); graphs (b) and (d) show mean values for protein levels±S.E. for 4–6 independent experiments, in each case normalized to ERK levels. (e and f) Trib3 contributes to the decrease in Akt phosphorylation that occurs after NGF deprivation. SCG neuron cultures 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 mentioned above. Panel (e) shows representative immunoblots and (f) shows mean values for protein levels±S.E. for 5–6 independent experiments, each normalized against total ERK. *P<0.05, **P<0.005; Student's paired t-test