Fig. 5.
CIRP enhances the binding of HuR to the cyclin E1 mRNA 3’UTR. A, MCF-7 cells were transfected with control siRNA (si-ctrl), CIRP siRNA (si-CIRP), pTracer-CMV2 vector (vec), or pTracer-CMV2-CIRP (CIRP). Equal amounts of cell lysates were incubated with 32P-labeled cyclin E1 mRNA 3’UTR, UV-crosslinked, and treated with RNase. The resulting RNA-protein complexes were precipitated with HuR antibody/protein A/G agarose and resolved on a 10% SDS-polyacrylamide gel. Arrow indicates radiolabeled protein immunoprecipitated by the HuR antibody. B, Gel shift assay. 32P-labeled cyclin E1 mRNA 3’UTR (1 fmol) was incubated with the indicated amounts of GST-HuR +/− 50 nM GST-CIRP. RNA-protein complexes were resolved on a 5% native polyacrylamide gel, phophorimaged and Kd calculated using a one-phase exponential decay. All experiments were repeated at least 3 times.