FIG. 3.

Selection of K-RBP binding sequences. (A) Schematic representation of the procedure used to identify K-RBP binding sequences. (B) Consensus (Con) sequences for K-RBP binding. Numbers indicate the frequencies of the specific nucleotide at each position (Pos). The most abundant nucleotides at each position were used to generate the consensus sequence shown in the bottom. The underlined CGG motif was pre-synthesized in the pool of the second round selection oligomers. (C) K-RBP binds clone #14 probe in EMSA. The probe was labeled with [α-³²P]dATP. The clone #14 and Oct-1 probe were used as unlabeled competitors. Unlabeled competitors were added at an 80–100-fold molar excess over the labeled probe. Various amounts of Ni-NTA agarose beads (5 and 15 μl) were used to remove the His-tagged K-RBP protein in EMSA. GST beads (15 μl) were used as a control. Mouse anti-His or Flag antibodies were added in the reaction prior to adding the probe as shown in lanes 11 and 12. The arrow indicates the bound complex. One μl K-RBP protein contains 5 ng purified K-RBP protein approximately.