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. 2014 Jul;20(7):1046–1056. doi: 10.1261/rna.044917.114

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© 2014 Kossinova et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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FIGURE 2. — CTSBP2-ribosome cross-linking using bifunctional reagents. (A) Distribution analysis of the diepoxybutane-induced SBP2-ribosome cross-links among ribosomal subunits isolated by sucrose gradient centrifugation under dissociation conditions. (1) CTSBP2 alone as the control; (2) 40S•CTSBP2 complex; (3) 60S•CTSBP2 complex; (4,5) 60S and 40S subunits, respectively, isolated from the 80S•CTSBP2 complex; (6) 60S•CTSBP2 complex purified by sucrose gradient under nondissociating conditions; (7) 60S•CTSBP2 complex untreated with diepoxybutane but purified by sucrose gradient under dissociation conditions. (B) Dot blot analysis of the SBP2-ribosome cross-link distribution among ribosomal proteins and rRNA; treatment with diepoxybutane (lane 1), 2-iminothiolane (lane 2), Control (lane 3): SBP2•ribosome complexes untreated with the bifunctional reagents. (C) Dot-blot analysis of diepoxybutane- (top) or 2-iminothiolane- (bottom) induced CTSBP2-rRNA cross-links. After chemical treatment, rRNAs were isolated from the 60S•CTSBP2 complexes by SDS-EDTA sucrose gradient; a sucrose gradient sedimentation profile is shown. Gradient fractions are indicated below the dot blots. CTSBP2 signals were obtained by dot blot analysis with anti-SBP2 antibodies.