Fig. 4.

Wig-1 binds to p53 mRNA in a U-rich region-dependent manner. (A) To establish whether Wig-1 binds directly to p53 mRNA, we performed a RNA IP experiment on lysates from cells transfected with FLAG-Wig-1 (Wig-1) or empty vector (FLAG). Wig-1 was precipitated with anti-FLAG beads, and bound RNA was purified and analyzed by real-time PCR. The graph shows the average of four independent experiments, error bars are shown with SEM, and asterisk indicates a statistically significant difference (P = 0.044). (B) Wig-1 protein levels were analyzed by Western blotting to ensure efficient pull-down. (C) To determine what region was required for Wig-1 binding to the p53 mRNA, we used the biotinylated p53 RNA probes to pull down Wig-1 from lysates of U2OS cells overexpressing FLAG-Wig-1. The constructs +U and −U differs in 70 nt containing the U-rich region. The probes were captured by streptavidin beads, and bound protein was analyzed by Western blotting (D).