HuR is a trans -factor binding in vitro to the HCE motif shared by mRNAs encoding RRM-type RBPs. The different RNA probes used for the protein pull-down experiment are shown in ( A ). HuR pull-down probe: this probe was designed by using the secondary structure motif reported in Figure 3 , slightly modifying the lowest part of the hairpin so as to make it fold correctly when not in context. The loop was designed by selecting the most probable bases of the sequence in the aligment and the most probable structure motifs. Positive controls probes are the known binding sites for the YB1 and PTB RBPs, experimentally obtained ( 11 ). Again, the lowest part of the stem was slightly modified so as to make it fold as desired. Negative controls HuR probes are Dbl-Mut1, Dbl-Mut2 and Degenerate. The Degenerate probe was synthesized by allowing all four nucleotides to be present at each loop position, so as to obtain a mixture of probes bearing all the possible eptameric loops. The Dbl-Mut1 and Dbl-Mut2 probes were obtained respectively by mutating two bases of the original probe loop in a way to preserve it and by mutating one base and deleting two others in a way to obtain a pentameric loop instead of a eptameric loop. In all probes, the 5′ circle represents the biotinylated DNA polyC linker. ( B ) shows the HuR pull-down western blots. From the leftmost band to the rightmost: Input, HuR probe, Dbl-Mut1, Dbl-Mut2, Degenerate probe and denaturated beads bands. As can be readily seen, the stem–loop probes bind to HuR with a marked specificity for the correct one. ( C and D ) YB1 and PTB RBPs pull-down. From the leftmost band to the rightmost: input, YB1/PTB probe and denaturated beads. As shown by western blotting, the stem–loop probes bind to PTB and YB1 respectively, thus confirming that the pull-down protocol works as expected.