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. 2009 Jun 8;37(14):4672–4683. doi: 10.1093/nar/gkp470

Figure 1.

Figure 1.

PTB binding to a pyrimidine tract within the HBB 3′ UTR is impaired by the β-thalassemia term+6 mutation. (a) Schematic representation of the 3′ terminal region of the HBB pre-mRNA showing the sequence of two pyrimidines tracts, PYR1 and PYR2, located at the last intron (IVSII) 3′ splice site (3′SS) and in the terminal exon (exon 3), respectively. The sequence difference between the PYR2 and term+6, which contains the β-thalassemia C to G mutation at 6 nucleotides after the stop codon (underlined), is shown. (b) UV crosslinking using 5′ 32P-labeled RNA oligonucleotides corresponding to the PYR2 or term+6 RNAs in the presence of NEs and resolution by SDS–PAGE. The 55-kDa protein UV crosslinked to the PYR2 but not to the term+6 RNA is indicated by the arrow. (c) IP of the UV-crosslinked complexes described in (b) with the PTB antibody (Bb7) followed by SDS–PAGE analysis. (d) UV crosslinking of GST-tagged PTB (2.5, 1.25 or 0.6 pmol) to the PYR2 or term+6 RNAs. Lanes 1 and 5: UV crosslinking in the absence of PTB. (e) IP following UV crosslinking of NEs to 5′ 32P-labeled PYR1 or PYR2 with the U2AF65 antibody followed by SDS–PAGE analysis.