Figure 5.

DANCR promotes translation of FOXO3 gene by binding to AUF1. (A) RIP was performed using anti-AUF1 and control IgG antibodies, followed by qRT-PCR to determine the enrichment of DANCR and U6. U6 was used as the negative control, ^**P<0.01. (B) Experimental validation of AUF1 knockdown in RLE-6TN cells by qRT-PCR and western blotting, ^***P<0.001. (C) mRNA and protein levels of FOXO3 in RLE-6TN cells silenced with AUF1. (D) Western blotting was performed to detect the protein expression levels of FOXO3 in RLE-6TN cells overexpressing DANCR or (and) silenced with AUF1. (E) RLE-6TN lysates were incubated with in vitro-synthesized, biotin-labeled control LacZ DNA probes or DNA probes against DANCR for the biotinylated oligonucleotide pulldown assay. ^**P<0.01 compared to respective LacZ probes. (F) Endogenous AUF1 binding to FOXO3 mRNA was modified by DANCR overexpression, ^**P<0.01. (G) Control plasmids or DANCR plasmids transfected RLE-6TN cells were cultured with 20 μg/ml cycloheximide (CHX) for 0-150 min and analyzed by western blotting.