Figure 3. HuR binds directly to the 5′ UTR of Bcl_xL and regulates its translation. (A) Schematic diagram of Bcl_xL 5′ UTR and the deletion fragments used in the binding assays. (B) Recombinant GST-HuR was incubated in the presence of ³²P-labeled, in vitro transcribed RNA (probe A) and subjected to UV cross-linking. RNA-protein complexes were separated by SDS-PAGE and analyzed by autoradiography. GST was used as a negative control. (C) Increasing concentrations of GST-HuR were incubated with indicated, ³²P-labeled, in vitro transcribed RNAs and nitrocellulose filter binding assays were performed as described in Material and Methods. Levels of filter-bound RNA are plotted as a function of protein concentration. Apparent dissociation constants (K_d) are shown for each probe (mean +/− S.E.M., n = 3). (D) Bicistronic DNA construct containing Bcl_xL 5′ UTR (pßgal/BclxL/CAT), or a parental vector (pßgal/CAT) were co-transfected into HEK293 cells along with GFP- or GFP-HuR expressing plasmids and the expression levels of each reporter gene were determined 24 h after transfection (** p < 0.01; n = 3).