CPEB controls the synthesis of Stat3 and PTEN.

<p>(A and B) 3′ UTR sequences of Stat3 and PTEN from human, mouse and cow. The nucleotides in bold represent putative CPEs. (C and D) Western blots of Stat3 and PTEN following CPEB depletion in HepG2 cells. In panels C-H, tubulin served as a negative or input control. (E and F) Quasi-quantitative RT-PCR for Stat3 and PTEN RNAs following CPEB depletion. (G and H) HepG2 cells depleted of CPEB were pulse labeled with ³⁵S-methionine for 15 min followed by Stat3, PTEN and tubulin (as a control) immunoprecipitation and SDS-PAGE analysis. These same proteins were also analyzed by western blots. (I) Representation of Renilla and firefly luciferase RNAs that were electroporated into HepG2 cells. Renilla luciferase RNA, which contained an irrelevant 3′ UTR, served as a normalization control. Firefly luciferase contained the Stat3 or PTEN 3′ UTRs as noted in panels A and B; in some cases, the CPEs in these 3′ UTRs were mutated. (J and K) The firefly and Renilla RNAs noted above were electroporated into HepG2 cells, some of which were depleted of CPEB. Firefly luciferase was normalized to the Renilla luciferase transfection control; luciferase activity derived from all RNAs was then made relative to the control shRNA. The Stat3 and PTEN data were analyzed with ANOVA; p values were 0.009 and 0.005, respectively. The asterisk refers to statistical significance (p<0.05). Data are represented as mean +/− SEM. The firefly and Renilla luciferase RNAs were also analyzed for relative stability by quasi-quantitative RT-PCR; all the RNAs had similar stabilities. At least 3 animals were used for each experiment.