Figure 6.

HuR interacts with the Cnpy3 mRNA and increases its stability and translation in cultured IECs. (A) Association of endogenous HuR with endogenous Cnpy3 mRNA as measured by RIP and RT-Q-PCR analysis using either anti-HuR antibody (Ab) or control IgG. Values are the means ± SEM from triplicate samples. *P < 0.05 compared with IgG IP. (B) HuR immunoblots using the materials pulled down by biotinylated transcripts of the Cnpy3 5’-UTR, CR, and 3’-UTR. Left, schematic representation of various biotinylated Cnpy3 transcripts. (C) Levels of the Cnpy3 mRNA at steady state (i) and in decay assays (ii) 48 h after transfection with C-siRNA and siHuR. Cnpy3 mRNA levels were examined at different times after administration of actinomycin D. *P < 0.05 compared with C-siRNA. (D) Newly synthesized CNPY3 protein as measured by L-azidohomoalaine (AHA) incorporation assays in cells described in C. (E) Distribution of Cnpy3 (top) and Gapdh (bottom) mRNAs in each gradient fraction of polysomal profiles prepared from cells described in C. (F) Levels of the Luc reporter activity 48 h after transfection with siHuR. Top, schematic of different chimeric firefly luciferase reporters bearing the Cnpy3 5’-UTR, CR, or 3’-UTR. *P < 0.05 compared with C-siRNA.