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. 2013 Apr 15;123(5):2103–2118. doi: 10.1172/JCI61820

Figure 5. Binding of biotin-labeled candidate mRNAs to PCBP2.

Figure 5

(A) Schematic of the 8 indicated candidate mRNAs. The specific fragments used as templates for the synthesis of biotin-labeled RNAs are indicated with underlines, and the nucleotide positions amplified by PCR are shown. (B) Biotin pull-down analysis of complexes formed in vitro using biotin-labeled candidate mRNA segments (shown in A) and T98G whole-cell lysates. The α-globin-3′ UTR and a nonsense sequence were included as the positive and negative controls, respectively. (C) A specific association between PCBP2 and its candidate target mRNAs (identified in B) was confirmed by a competition assay. Five- or 10-fold excess of unlabeled RNA was added to compete with the biotin-labeled RNA for interaction with PCBP2, and 2- or 3-fold excess of cell lysates was incubated with biotin-labeled RNA. (D) Detection of the binding affinities of the target mRNAs to PCBP2 by biotin pull-down analysis using all of the biotin-labeled RNAs identified above.