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. 2021 Jun 9;49(11):6456–6473. doi: 10.1093/nar/gkab484

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© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — RP-CONA detects the specific interaction of pre-let-7a-1-CTL/Lin28a. (A) A diagram illustrating the interaction between Cy5-pre-let-7a-1-CTL and Lin28a-GFP. (B) Beads images of Cy5-pre-let7a-1-CTL-beads incubated in lysates-free buffer; blank beads incubated with cell lysates containing Lin28a-GFP; Cy5-pre-let7a-1-CTL-beads incubated with cell lysates containing Lin28a-GFP; and Cy5-pre-let-7a-1-CTL-beads incubated with cell lysates containing GFP. (C andD) GFP/Cy5 signals were reduced by unlabelled pre-let-7a-1. Cell extracts containing 300 nM of GFP-Lin28a were treated with 80 pmol of pre-let-7a-1 or pre-let-7a-1/miR-16-TL, prior to pull-down with Cy5-pre-let7a-1-CTL-beads. (E andF). GFP/Cy5 signals were reduced by anti-Lin28a antibody. Cell extracts containing 300 nM Lin28a-GFP were pre-incubated with 2 μl (+) or 4 μl (++) of anti-HuR antibody (control) or anti-Lin28a antibody prior pulldown by Cy5-pre-let-7a-1-CTL-beads. Mean GFP/Cy5 ring intensities and SD between triplicates are shown. Statistically significant differences compared to HuR antibody treated samples were interpreted by SPSS independent sample t-test, ***P< 0.001, ****P< 0.0001.