CPEB depletion in HepG2 cells results in aberrant insulin signaling.

(A) HepG2 cells were infected with lentiviruses expressing a control shRNA or one directed against CPEB; extracted RNA was then assessed for CPEB RNA, and tubulin as a control. (B) HepG2 cells were co-infected with the lentiviruses noted above as well as a retrovirus expressed FLAG-CPEB; western blots were probed for the FLAG epitope and tubulin. (C) HepG2 cells infected with lentivirus expressing control or CPEB shRNA were treated with insulin and western blotted for phospho (S473 and T308) and total Akt. The pAkt (Ser473) and pAkt (Thr308) data were analyzed with ANOVA (p<0.05, *). All experiments were performed 3 times. (D) HepG2 cells co-infected with the viruses noted above were western blotted for phospho (S473) and total Akt. The pAkt (Ser473) data was analyzed by ANOVA (p<0.05). (E) HepG2 cells infected with virus expressing FLAG-CPEB or FLAG-CPEBΔZF, which lacks two zinc fingers and cannot bind RNA, followed by FLAG co- immunoprecipitation and analysis of Stat3, PTEN, PDK1, IRS1, IRS2, and Socs3 RNAs by quasi-quantitative RT-PCR. Input represents 10% of total. At least 3 animals per group were used for the experiment.