Fig. 3.

CIRP and HuR bind the cyclin E1 mRNA. A, UV-crosslink competition analysis. An equal amount of GST-CIRP or GST-HuR was incubated with ³²P-labeled cyclin E1 mRNA 3’UTR (E13’UTR) in the presence of 0 (lanes 1 and 6) or 100 molar excess unlabeled specific (cold E13’UTR, lanes 2 and 7) or nonspecific (cold E1CR378, lanes 3 and 8) competitor. Arrows indicate protein binding to the cyclin E1 3’UTR. Lane 4, radiolabeled RNA alone; lane 5, radiolabeled RNA treated with RNase in the absence of added protein. B, UV-crosslink immunoprecipitation analysis. MCF-7 cell extract was incubated with ³²P-labeled cyclin E1 mRNA 3’UTR (E13’UTR) or cyclin E1CR378 mRNA. After crosslinking and RNase treatment, RNA-protein complexes were precipitated with CIRP antibody (α-CIRP) or normal IgG followed by protein A/G agarose beads and resolved on a 12% SDS-polyacrylamide gel. Input lane is the UV-crosslinked ³²P-labeled cyclin E1 3’UTR with MCF-7 cell extract. Arrow indicates the 18-kDa protein immunoprecipitated by CIRP antibody. Experiments were repeated at least 3 times.