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. 2016 Oct 26;291(50):25823–25836. doi: 10.1074/jbc.M116.756908

FIGURE 5.

FIGURE 5.

AUF1 interacts with ATX 3′UTR and destabilizes ATX mRNA. A and B, the effects of AUF1 knockdown and overexpression on ATX expression. Colo320 cells were transfected with the AUF1 siRNA (A) or with the plasmid expressing AUF1 (pcDNA3.1-AUF1) (B), respectively. At 48 h after the transfection, the protein levels of AUF1 in cell lysate and ATX in culture medium by Western blotting analysis, and RNA isolated from the cells were subjected to RT-qPCR to assess the ATX mRNA levels. C, the Colo320 cell lysates were incubated with each of the biotinylated ATX mRNA fragments as indicated. After biotin pulldown, AUF1 bound to the ATX mRNA fragments was detected by Western blotting. D and E, the effects of AUF1 knockdown and overexpression on ATX mRNA stability. Colo320 cells were transfected with the AUF1-specific siRNA (D) or with the plasmid expressing AUF1 (pcDNA3.1-AUF1) (E), respectively. At 48 h after the transfection, the cells were exposed to actinomycin D (1 μg/ml). The cellular RNA was isolated at the time points after actinomycin D addition as indicated, and real-time RT-qPCR analyses were performed to assess the half-lives of ATX mRNA. *, p < 0.05, **, p < 0.01.