FIGURE 7.

AUF1 binds and stabilizes the PDK1 mRNA. A, whole cell lysates were prepared from the indicated cells and used for immunoblotting analysis utilizing antibodies against the indicated proteins. B, total RNA was prepared from the indicated cells and utilized to amplify the indicated transcripts by qRT-PCR using specific primers. C, U2OS and EH1 cells expressing the indicated constructs were treated with actinomycin D and then reincubated for the indicated periods of time. Total RNA was extracted, and the remaining amount of the PDK1 mRNA was assessed using qRT-PCR. The dashed lines indicate the PDK1 mRNA half-life. Error bars, S.E. values of three different experiments. D, biotinylated PDK1 3′-UTR bearing either wild type or mutated sequence of the AUF1 binding site was incubated with cytoplasmic cellular lysate from the indicated cells, and the association of AUF1 with these RNAs was detected by immunoblotting using anti-AUF1 antibody. E, U2OS cells expressing AUF1 siRNA or a control plasmid were stably transfected with the luciferase reporter vector bearing either the wild-type PDK1 3′-UTR or a mutated sequence for the binding site of AUF1 (residues 556–562). The reporter activity was assessed at 48 h post-transfection. Data (mean ± S.E., n = 4) were presented as a percentage change in reporter activity as compared with the negative control cells (*) or with the wild-type 3′-UTR (**). * and **, p < 0.00003.