Figure 6.

HuR binds to Cox-2 mRNA, contributing to the sustained induction of Cox-2 by LPA. A) Binding of HuR with the 3′UTR of Cox-2 mRNA in vitro. In vitro transcribed, biotinylated 3′UTR of the Cox-2 mRNA (1.5 μg) was incubated with cellular protein (90 μg) from Caov-3 cells treated with BSA or LPA (10 μM, 6 h). Amounts of HuR pulled down by the Cox-2 3′UTR were analyzed by immunoblotting with an anti-HuR antibody. Cell lysates (30 μg) were included to serve as inputs. B) Association of HuR protein with Cox-2 mRNA within the cell. HuR was immunoprecipitated from Caov-3 cells treated for 6 h with BSA (control) or LPA (10 μM). The mRNAs present in the HuR immunocomplex were extracted, reverse-transcribed, and amplified by PCR using Cox-2 specific primers as detailed in Materials and Methods. C) Effect of HuR knockdown on LPA-induced Cox-2 expression. The control and HuR down-regulated Caov-3 cells were stimulated for 6 h (left panel) or 12 h (right panel) with 10 μM LPA. Cox-2, Cox-1 and HuR levels in these cells were analyzed with immunoblotting. The values beneath each lane represent relative intensities (%) quantified by densitometry, with Cox-2 induced by LPA in control siRNA-treated cells defined as 100%. Similar results were obtained from two independent assays.